Cyclic depsipeptides

ABSTRACT

The present application relates to cyclic depsipeptides, or derivatives thereof, having the structure of formula (I), and uses thereof, e.g. as inhibitors of kallikrein 7 and human neutrophil elastase.

FIELD OF THE INVENTION

The present invention relates to cyclic depsipeptides, or a derivativesthereof.

BACKGROUND OF THE INVENTION

Kallikrein 7 is a S1 serine protease of the kallikrein gene familydisplaying a chymotrypsin like activity. Human kallikrein 7 (hK7, KLK7or stratum corneum chymotryptic enzyme (SCCE), Swissprot P49862) playsan important role in skin physiology (1, 2, 3). It is mainly expressedin the skin and has been reported to play an important role in skinphysiology. hK7 is involved in the degradation of the intercellularcohesive structures in cornified squamous epithelia in the process ofdesquamation. The desquamation process is well regulated and delicatelybalanced with the de novo production of corneocytes to maintain aconstant thickness of the stratum corneum, the outermost layer of theskin critically involved in skin barrier function. In this regard, hK7is reported to be able to cleave the corneodesmosomal proteinscorneodesmosin and desmocollin 1 (4, 5, 6). The degradation of bothcorneodesmosomes is required for desquamation. In addition, veryrecently it has been shown that the two lipid processing enzymesβ-glucocerebrosidase and acidic sphingomyelinase can be degraded by hK7(7). Both lipid processing enzymes are co-secreted with their substratesglucosylceramides and sphingomyelin and process these polar lipidprecursors into their more non-polar products e.g. ceramides, which aresubsequently incorporated into the extracellular lamellar membranes. Thelamellar membrane architecture is critical for a functional skinbarrier. Finally, hK7 has been shown to activate Interleukin-1β (IL-1β)precursor to its active form in vitro (8). Since keratinocytes expressIL-1β but not the active form of the specific IL-1β converting enzyme(ICE or caspase 1), it is proposed that IL-1β activation in humanepidermis occurs via another protease, a potential candidate being hK7.

Recent studies link an increased activity of hK7 to inflammatory skindiseases like atopic dermatitis, psoriasis or Netherton's syndrome. Thismight lead to an uncontrolled degradation of corneodesmosomes resultingin a miss-regulated desquamation, an enhanced degradation of lipidprocessing enzymes resulting in a disturbed lamellar membranearchitecture or an uncontrolled activation of the proinflammatorycytokine IL-1β. The net result would be an impaired skin barrierfunction and inflammation (see also WO-A-20041108139).

Due to the fact that the hK7 activity is controlled at several levels,various factors might be responsible for an increased hK7 activity ininflammatory skin diseases. Firstly, the amount of protease beingexpressed might be influenced by genetic factors. Such a genetic link, apolymorphism in the 3′-UTR in the hK7 gene, was recently described (9).The authors hypothesise that the described 4 base pair insertion in the3′-UTR of the kallikrein 7 gene stabilizes the hK7 mRNA and results inan overexpression of hK7. Secondly, since hK7 is secreted via lamellarbodies to the stratum corneum extracellular space as zymogen and it isnot able to autoactivate, it needs to be activated by another proteasee.g. hK5 (5). Uncontrolled activity of such an activating enzyme mightresult in an overactivation of hK7. Thirdly, activated hK7 can beinhibited by natural inhibitors like LEKTI, ALP or elafin (10, 11). Thedecreased expression or the lack of such inhibitors might result in anenhanced activity of hK7. Recently it was found, that mutations in thespink5 gene, coding for LEKTI, are causative for Netherton's syndrome(12) and a single point mutation in the gene is linked to atopicdermatitis (13, 14). Finally, another level of controlling the activityof hK7 is the pH. hK7 has a neutral to slightly alkaline pH optimum (2)and there is a pH gradient from neutral to acidic from the innermost tothe outermost layers in the skin. Environmental factors like soap mightresult in a pH increase in the outermost layers of the stratum corneumtowards the pH optimum of hK7 thereby increasing the hK7 activity.

The hypothesis that an increased activity of hK7 is linked to skindiseases with an impaired skin barrier including inflammatory andhyperpoliferative skin diseases is supported by the following studies:Firstly, Netherton's syndrome patients show a phenotype dependentincrease in serine protease activity, a decrease in corneodesmosomes, adecrease in the lipid processing enzymes β-glucocerebrosidase and acidicsphingomyelinase, and an impaired barrier function (15, 16). Secondly, atransgenic mice overexpressing human kallikrein 7 shows a skin phenotypesimilar to that found in patients with atopic dermatitis (17, 18, 19).Thirdly, in the skin of atopic dermatitis and psoriasis patientselevated levels of hK7 were described (17, 20). Furthermore, increasedactivity of K₇ and thus epithelial barrier dysfunction may also play animportant role in the pathology of other epithelial diseases such asinflammatory bowel disease and Crohn's disease.

Therefore, hK7 is considered to be a potential target for the treatmentof diseases involved with epithelial dysfunction such as inflammatoryand/or hyperpoliferative and pruritic skin diseases like atopicdermatitis, psoriasis, Netherton's syndrome or other pruritic dermatosessuch as prurigo nodularis, unspecified itch of the elderly as well asother diseases with epithelial barrier dysfunction such as inflammatorybowel disease and Crohn's disease and there is a need for specificmodulators (agonists or inhibitors) thereof.

Human neutrophil elastase (HNE, also know as human leukocyte elastase,HLE) belongs to the chymotrypsin family of serine proteinases. Itscatalytic activity is optimal around pH 7, and the catalytic site iscomposed of three hydrogenbonded amino acid residues: His57, Asp102, andSer195 (in chymotrypsin numbering), which form the so-called catalytictriad. The enzyme is composed of a single peptide chain of 218 aminoacid residues and four disulfide bridges. It shows 30 to 40% sequenceidentity with other elastinolytic or nonelastinolytic serineproteinases. HNE preferentially cleaves the oxidized insulin B chainwith Val at the P1 position, but it also hydrolyzes bonds with Ala, Ser,or Cys in the P1 position.

HNE is located in the azurophilic granules of polymorphonuclearleukocytes (PMNLs), where the HNE concentration is rather high (3 μg ofenzyme/106 cells). The major physiological function is to digestbacteria and immune complexes and to take part in the host defenseprocess. HNE aids in the migration of neutrophils from blood to varioustissues such as the airways in response to chemotactic factors. HNE alsotakes part in wound healing, tissue repair, and in the apoptosis ofPMNLs.

In addition to elastin (highly flexible and highly hydrophobic componentof lung connective tissue, arteries, skin, and ligaments), HNE cleavesmany proteins with important biological functions, including differenttypes of collagens, membrane proteins, and cartilage proteoglycans. HNEalso indirectly favours the breakdown of extracellular matrix proteinsby activating procollagenase, prostromelysin, and progelatinase. HNEinactivates a number of endogenous proteinase inhibitors such asα2-antiplasmin, α1-antichymotrypsin, antithrombin, and tissue inhibitorof metalloproteinases.

Extracellular elastase activity is tightly controlled in the pulmonarysystem by α1-protease inhibitor (α1PI), responsible for protection ofthe lower airways from elastolytic damage, whereas the secretoryleukocyte proteinase inhibitor protects mainly the upper airways. In anumber of pulmonary pathophysiological states, e.g., pulmonaryemphysema, chronic bronchitis, and cystic fibrosis, endogenous elastaseinhibitors are inefficient in regulating HNE activity.

HNE is considered to be the primary source of tissue damage associatedwith inflammatory diseases such as pulmonary emphysema, adultrespiratory distress syndrome (ARDS), chronic bronchitis, chronicobstructive pulmonary disease (COPD), pulmonary hypertension, and otherinflammatory diseases as well as bronchopulmonary dysplasia in prematureneonates. HNE is involved in the pathogenesis of increased and abnormalairway secretions commonly associated with airway inflammatory diseases.Thus, bronchoalveolar lavage (BAL) fluid from patients with chronicbronchitis and cystic fibrosis has increased HNE activity. Furthermore,excessive elastase has been proposed to contribute not only to thesechronic inflammatory diseases but also to acute inflammatory diseasessuch as ARDS and septic shock.

Therefore, HNE is considered to be a potential target for the treatmentof diseases involved with HNE activity such as inflammatory diseasessuch as pulmonary emphysema, adult respiratory distress syndrome (ARDS),chronic bronchitis, chronic obstructive pulmonary disease (COPD),pulmonary hypertension, and other inflammatory diseases as well asbronchopulmonary dysplasia in premature neonates, and diseases involvedwith increased and abnormal airway secretions as well as acuteinflammatory diseases. Thus there is a need for specific modulators(agonists or inhibitors) if HNE.

Treatment can be by local or systemic application such a creams,ointments and suppositories or by oral or sc or iv application or byinhalation, respectively.

Chondromyces is a genus in the family Polyangiaceae, which belongs tothe order Myxococcales within the Delta-proteobacteria. Bacteria of theorder Myxococcales, also called myxobacteria, are gram-negativerod-shaped bacteria with two characteristics distinguishing them frommost other bacteria. They swarm on solid surfaces using an activegliding mechanism and aggregate to form fruiting bodies upon starvation(Kaiser (2003)). The present inventors have identified cyclicdepsipeptide produced by Chondromyces that are able to specificallymodulate kallikrein 7.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to cyclic depsipeptides, orderivatives thereof, having the structure of formula (I):

wherein the ester bond is found between the carboxy group of A7 and thehydroxy group of A2, wherein X and A₁ are each independently optional,and wherein X is any chemical residue, wherein A₁ is a standard aminoacid which is not aspartic acid or a derivative of said standard aminoacid, wherein A₂ is threonine or serine, wherein A₃ is a non-basicstandard amino acid or a non-basic derivative thereof, wherein A₄ isAhp, dehydro-AHP, proline or a derivative thereof, wherein A₅ isisoleucine or valine, wherein A₆ is tyrosine or a derivative thereof andwherein A₇ is leucine, isoleucine or valine.

Alternatively, the cyclic depsipeptides of the invention, or derivativesthereof, can be depicted according to Formula (I′):

wherein X and A₁ are as defined in the embodiments, and whereinR2 is H or methylR3 the side chain of a non-basic amino acid or a non-basic derivativethereofR5 is the side chain of the amino acid isoleucine or valineR6 is the side chain of tyrosine or a derivative thereofR7 is the side chain of the amino acid leucine, isoleucine or valineY is either hydrogen or a methylgroup,and wherein Ahp can be substituted by dehydro-AHP, Ahp-I, Ahp-II,proline or a derivative thereof.

The present invention also relates to a pharmaceutically acceptable saltof such a cyclic depsipeptide or a derivative thereof.

In the cyclic depsipeptides of the invention X can be H or an acylresidue, for instance CH₃CH₂CH(CH₃)CO, (CH₃)₂CHCH₂CO or (CH₃)₂CHCO.

In the cyclic depsipeptides of the invention A1 can be glutamine,glutamic acid, or a derivative thereof, e.g. a glutamic nitrile or aglutamic acid ester.

In the cyclic depsipeptides of the invention A2 can be threonine or aderivative thereof.

In the cyclic depsipeptides of the invention A3 can be leucine.

In the cyclic depsipeptides of the invention A6 can be tyrosine.

In some embodiments of the cyclic depsipeptides of the invention A4 canbe the Ahp derivative 3-amino-piperidin-2-one, Ahp-I or Ahp-H.

In some embodiments of the cyclic depsipeptides, or derivatives thereof,of the invention, X is (CH₃)₂CHCO, A₁ is glutamine, glutamic acid or aderivative thereof, A₂ is threonine, A₃ is leucine, A₄ is Ahp or aderivative thereof, A₅ is isoleucine or valine, A₆ is tyrosine or aderivative thereof and A₇ is isoleucine or valine.

In other embodiments of cyclic depsipeptide, or derivatives thereof ofthe invention X is CH₃CH₂CH(CH₃)CO A₁ is glutamine, glutamic acid or aderivative thereof A₂ is threonine, A₃ is leucine, A₄ is Ahp or aderivative thereof, A₅ is isoleucine, A₆ is tyrosine or a derivativethereof and A₇ is isoleucine.

In yet other embodiments of cyclic depsipeptide, or derivative thereof,of the invention X is CH₃CH₂CH(CH₃)CO, A₁ is glutamine, glutamic acid ora derivative thereof, A₂ is threonine, A₃ is leucine, A₄ is dehydro-AHPor a derivative thereof, A₅ is isoleucine, A₆ is tyrosine or aderivative thereof, and A₇ is isoleucine.

In further embodiments of cyclic depsipeptide, or derivative thereof, ofthe invention X is (CH₃)₂CHCH₂CO, A₁ is glutamine, glutamic acid or aderivative thereof, A₂ is threonine, A₃ is leucine, A₄ is dehydro-AHP,Ahp or a derivative thereof, A₅ is isoleucine, A₆ is tyrosine or aderivative thereof, and A₇ is isoleucine.

The present invention moreover also relates to cyclic depsipeptides, orderivatives thereof, having the structure of formula (I)

wherein the ester bond is found between the carboxy group of A7 and thehydroxy group of A2, wherein X is (CH₃)₂CHCH₂CO, wherein A₁ isglutamine, glutamic acid or a derivative thereof, wherein A₂ isthreonine, wherein A₃ is leucine, wherein A₄ is Ahp or proline, or aderivative thereof, wherein A₅ is phenylalanine, wherein A₆ is tyrosineor a derivative thereof, and wherein A₇ is valine.

In particular embodiments thereof, X is (CH₃)₂CH CH₂CO, A₁ is glutamine,glutamic acid or a derivative thereof, A₂ is threonine, A₃ is leucine,A₄ is Ahp, or a derivative thereof, A₅ is phenylalanine, A₆ is tyrosineor a derivative thereof, and A₇ is valine.

In other embodiments thereof, X is (CH₃)₂CH CH₂CO, A₁ is glutamine,glutamic acid or a derivative thereof A₂ is threonine, A₃ is leucine, A₄is proline, or a derivative thereof, A₅ is phenylalanine, A₆ is tyrosineor a derivative thereof, and A₇ is valine. In there embodiments, thenitrogen atom of the amid bond between A5 and A6 can be substituted witha methyl.

In the cyclic depsipeptide, or derivative thereof, of the invention A1,A2, A3, A5, A6 and A7 can be L-amino acids. Moreover, A4 can 3S,6R Ahp.

The present invention also relates to the use of the above-describeddepsipeptides, and derivatives thereof, as a medicament. For instancefor the treatment of cancer, in particular ovarian cancer, or for thetreatment of inflammatory and/or hyperpoliferative and pruritic skindiseases such as keloids, hypertrophic scars, acne, atopic dermatitis,psoriasis, pustular psoriasis, rosacea, Netherton's syndrome or otherpruritic dermatoses such as prurigo nodularis, unspecified itch of theelderly as well as other diseases with epithelial barrier dysfunctionsuch as aged skin, inflammatory bowel disease and Crohn's disease, aswell as pancreatitis, or of cancer, in particular ovarian cancer, cysticfibrosis (CF), chronic obstructive pulmonary disease (COPD), pulmonaryfibrosis, adult respiratory distress syndrome, chronic bronchitis,hereditary emphysema, rheumatoid arthritis, IBD, psoriasis, asthma.

In one embodiment the present invention relates to the use of theabove-described depsipeptides, and derivatives thereof, as a medicamentfor the treatment of inflammatory and/or hyperpoliferative and pruriticskin diseases such as keloids, hypertrophic scars, acne, atopicdermatitis, psoriasis, pustular psoriasis, rosacea, Netherton's syndromeor other pruritic dermatoses such as prurigo nodularis, unspecified itchof the elderly as well as other diseases with epithelial barrierdysfunction such as aged skin, inflammatory bowel disease and Crohn'sdisease, as well as pancreatitis, or of cancer, in particular ovariancancer.

In another embodiment the present invention relates to the use of theabove-described depsipeptides, and derivatives thereof, as a medicamentfor the treatment of cystic fibrosis (CF), chronic obstructive pulmonarydisease (COPD), pulmonary fibrosis, adult respiratory distress syndrome,chronic bronchitis, hereditary emphysema, rheumatoid arthritis, IBD,psoriasis, asthma.

In yet another embodiment the present invention relates to the use ofthe above-described depsipeptides, and derivatives thereof, as amedicament for the treatment of inflammatory and/or hyperpoliferativeand pruritic skin diseases such as keloids, hypertrophic scars, acne,atopic dermatitis, psoriasis, pustular psoriasis, rosacea, Netherton'ssyndrome or other pruritic dermatoses such as prurigo nodularis,unspecified itch of the elderly.

The present invention also encompasses processes for producing thecyclic depsipeptide, or derivative thereof, of the invention, forinstance by cultivation of a Chondromyces strain, a variant or a mutantthereof, in a suitable medium, and optionally chemical derivation of theso-produced cyclic depsipeptide, or by expression of the biosynthesisgenes of a Chondromyces strain, a variant or a mutant thereof, in aheterologous microbial host strain, and optionally chemical derivationof the so-produced cyclic depsipeptide.

These processes of the invention can be performed with the strainsChondromyces crocatus (DSM 19329) or Chondromyces robustus (DSM 19330)or Chondromyces apiculatus (DSM 21595).

The present invention hence also relates to isolated Chondromycesmicroorganisms deposited under the accession number DSM 19329 or DSM19330 or DSM 21595 and to cyclic depsipeptides, or derivative thereof,produced by these isolated Chondromyces microorganisms.

DESCRIPTION OF THE FIGURES

FIG. 1: ¹H-NMR spectrum of the compound of formula (II) (600 MHz,d₆-DMSO)

FIG. 2: ¹³C-NMR spectrum of the compound of formula (II) (150 MHz,d₆-DMSO)

FIG. 3: ¹H-NMR spectrum of the compound of formula (VIII) (600 MHz,d₆-DMSO)

FIG. 4: ¹³C-NMR spectrum of the compound of formula (VIII) (150 MHz,d₆-DMSO).

FIG. 5: ¹H-NMR spectrum of a. derivative of the cyclic depsipeptideaccording to formula (II) wherein the Ahp has been converted into3-amino-piperidin-2-one (Example 4).

FIG. 6: ¹H NMR spectrum of a. derivative of the cyclic depsipeptideaccording to Example 5. (500 MHz, d₆-DMSO)

FIG. 7: ¹H-NMR spectrum of a. derivative of the cyclic depsipeptideaccording to Example 19. (500 MHz, d₆-DMSO)

FIG. 8: ¹H-NMR spectrum of a. derivative of the cyclic depsipeptideaccording to Example 21. (500 MHz, d₆-DMSO)

FIG. 9: ¹H NMR spectrum of a. derivative of the cyclic depsipeptideaccording to Example 26. (500 MHz, d₆-DMSO)

FIG. 10: ¹H-NMR spectrum of a. derivative of the cyclic depsipeptideaccording to Example 32. (500 MHz, d₆-DMSO)

FIG. 11: ¹H-NMR spectrum of a. derivative of the cyclic depsipeptideaccording to Example 44. (500 MHz, d₆-DMSO)

FIG. 12: ¹H-NMR spectrum of a. derivative of the cyclic depsipeptideaccording to Example 45 (500 MHz, d₆-DMSO)

DETAILED DESCRIPTION OF THE INVENTION

As described herein-above and in the embodiments, the present inventionrelates in one aspect to cyclic depsipeptides, or a derivatives thereof,having the structure of formula (I):

wherein the ester bond is found between the carboxy group of A7 and thehydroxy group of A2, wherein X and A₁ are each independently optional,and wherein X is any chemical residue, wherein A₁ is a standard aminoacid which is not aspartic acid, wherein A₂ is threonine or serine,wherein A₃ is a non-basic standard amino acid or a non-basic derivativethereof, wherein A₄ is Ahp, dehydro-AHP, proline or a derivativethereof, wherein A₅ is isoleucine or valine, wherein A₆ is tyrosine or aderivative thereof and wherein A₇ is leucine, isoleucine or valine.

Alternatively, the cyclic depsipeptides of the invention, or aderivative thereof, can be depicted according to Formula (I′):

wherein X and A₁ are as defined in the embodiments, and whereinR2 the side chain of the amino acid threonine or serineR3 the side chain of a non-basic amino acid or a non-basic derivativethereofR5 is the side chain of the amino acid isoleucine or valineR6 is the side chain of tyrosine or a derivative thereofR7 is the side chain of the amino acid leucine, isoleucine or valineY is either hydrogen or a methylgroup,and wherein Ahp can be substituted by dehydro-AHP, Ahp-I, Ahp-II,proline or a derivative thereof.

The present invention also relates to a pharmaceutically acceptable saltof such a cyclic depsipeptide or a derivative thereof.

In the cyclic depsipeptides of the invention the nitrogen atom of theamid bond between A5 and A6 can be substituted with a methyl.

In the cyclic depsipeptides of the invention X can be H or an acylresidue, for instance CH₃CH₂CH(CH₃)CO, (CH₃)₂CHCH₂CO or (CH₃)₂CHCO.

In the cyclic depsipeptides of the invention A1 can be glutamine,glutamic acid or a derivative thereof.

In the cyclic depsipeptides of the invention A2 can be threonine.

In the cyclic depsipeptides of the invention A3 can be leucine.

In the cyclic depsipeptides of the invention A6 can be tyrosine or aderivative thereof.

In some embodiments of the cyclic depsipeptides of the invention A4 canbe the Ahp derivative 3-amino-piperidin-2-one, Ahp-I or Ahp-H.

In some embodiments of cyclic depsipeptide, or derivative thereof, ofthe invention, X is (CH₃)₂CHCO, A₁ is glutamine, glutamic acid or aderivative thereof, A₂ is threonine, A₃ is leucine, A₄ is Ahp or aderivative thereof, A₅ is isoleucine or valine, A₆ is tyrosine or aderivative thereof and A₇ is isoleucine or valine.

In other embodiments of cyclic depsipeptide, or derivative thereof, ofthe invention X is CH₃CH₂CH(CH₃)CO A₁ is glutamine, glutamic acid or aderivative thereof, A₂ is threonine, A₃ is leucine, A₄ is Ahp or aderivative thereof, A₅ is isoleucine, A₆ is tyrosine or a derivativethereof, and A₇ is isoleucine.

In yet other embodiments of cyclic depsipeptide, or derivative thereof,of the invention X is CH₃CH₂CH(CH₃)CO, A₁ is glutamine, glutamic acid ora derivative thereof, A₂ is threonine, A₃ is leucine, A₄ is dehydro-AHPor a derivative thereof, A₅ is isoleucine, A_(s) is tyrosine or aderivative thereof, and A₇ is isoleucine.

In further embodiments of cyclic depsipeptide, or derivative thereof, ofthe invention X is (CH₃)₂CHCH₂CO, A₁ is glutamine, glutamic acid or aderivative thereof, A₂ is threonine, A₃ is leucine, A₄ is dehydro-AHP ora derivative thereof, A₅ is isoleucine, A₆ is tyrosine or a derivativethereof, and A₇ is isoleucine.

The present invention moreover also relates to cyclic depsipeptides, orderivatives thereof, having the structure of formula (I)

wherein the ester bond is found between the carboxy group of A7 and thehydroxy group of A2, wherein X is (CH₃)₂CHCH₂CO, wherein A₁ isglutamine, glutamic acid or a derivative thereof, wherein A₂ isthreonine, wherein A₃ is leucine, wherein A₄ is Ahp or proline, or aderivative thereof, wherein A₅ is phenylalanine, wherein A₆ is tyrosineor a derivative thereof, and wherein A₇ is valine.

In particular embodiments thereof, X is (CH₃)₂CH CH₂CO, A₁ is glutamine,glutamic acid or a derivative thereof, A₂ is threonine, A₃ is leucine,A₄ is Ahp, or a derivative thereof, A₅ is phenylalanine, A₆ is tyrosineor a derivative thereof, and A₇ is valine.

In other embodiments thereof, X is (CH₃)₂CH CH₂CO, A₁ is glutamine,glutamic acid or a derivative thereof A₂ is threonine, A₃ is leucine, A₄is proline, or a derivative thereof, A₅ is phenylalanine, A₆ is tyrosineor a derivative thereof, and A₇ is valine. In there embodiments, thenitrogen atom of the amid bond between A5 and A6 can be substituted witha methyl.

In the cyclic depsipeptide, or derivative thereof, of the invention A1,A2, A3, A5, A6 and A7 can be L-amino acids. Moreover, A4 can 3S,6R Ahp.

A5 stands for isoleucine, phenylalanine or valine. A5 is in particularisoleucine or valine, and preferably isoleucine.

In another embodiment, A5 may be phenylalanine in particular when A4 isnot Ahp, and the remaining variables are as defined in embodiment 1.

In another embodiment, A4 is 5-hydroxyproline and A5 is isoleucine, andthe remaining variables are as defined in embodiment 1.

In another embodiment, A4 is Ahp and A5 is isoleucine, and the remainingvariables are as defined in embodiment 1.

In another embodiment, A4 is Ahp-I and A5 is isoleucine, and theremaining variables are as defined in embodiment 1.

In another embodiment, A4 is Ahp-II and A5 is isoleucine, and theremaining variables are as defined in embodiment 1.

In another embodiment, A4 is Ahp, A5 and A7 is isoleucine, and theremaining variables are as defined in embodiment 1.

In another embodiment, A4 is Ahp-I, A5 and A7 is isoleucine, and theremaining variables are as defined in embodiment 1.

In another embodiment, A4 is Ahp-H, A5 and A7 is isoleucine, and theremaining variables are as defined in embodiment 1.

In another embodiment, A4 is 5-hydroxyproline, A5 and A7 is isoleucine,and the remaining variables are as defined in embodiment 1.

As used herein, A1 is a glutamine, glutamic acid, ornithine, or aglutamine derivative such as for example glutamic nitrile, glutamic acidester such as C₁₋₁₂alkyl ester (e.g. glutamic acid methyl ester) or suchas C₆₋₂₄aryl ester (e.g. glutamic acid phenyl or benzyl ester).

The present invention also relates to the use of the above-describeddepsipeptides, and derivatives thereof, as a medicament. For instancefor the treatment of cancer, in particular ovarian cancer, or for thetreatment of inflammatory and/or hyperpoliferative and pruritic skindiseases such as keloids, hypertrophic scars, acne, atopic dermatitis,psoriasis, Netherton's syndrome or other pruritic dermatoses such asprurigo nodularis, unspecified itch of the elderly as well as otherdiseases with epithelial barrier dysfunction such as inflammatory boweldisease and Crohn's disease, as well as pancreatitis.

In one embodiment the present invention relates to the use of theabove-described depsipeptides, and derivatives thereof, as a medicamentfor the treatment of inflammatory and/or hyperpoliferative and pruriticskin diseases such as keloids, hypertrophic scars, acne, atopicdermatitis, psoriasis, pustular psoriasis, rosacea, Netherton's syndromeor other pruritic dermatoses such as prurigo nodularis, unspecified itchof the elderly as well as other diseases with epithelial barrierdysfunction such as aged skin, inflammatory bowel disease and Crohn'sdisease, as well as pancreatitis, or of cancer, in particular ovariancancer.

In another embodiment the present invention relates to the use of theabove-described depsipeptides, and derivatives thereof, as a medicamentfor the treatment of cystic fibrosis (CF), chronic obstructive pulmonarydisease (COPD), pulmonary fibrosis, adult respiratory distress syndrome,chronic bronchitis, hereditary emphysema, rheumatoid arthritis, IBD,psoriasis, asthma. In yet another embodiment the present inventionrelates to the use of the above-described depsipeptides, and derivativesthereof, as a medicament for the treatment of inflammatory and/orhyperpoliferative and pruritic skin diseases such as keloids,hypertrophic scars, acne, atopic dermatitis, psoriasis, pustularpsoriasis, rosacea, Netherton's syndrome or other pruritic dermatosessuch as prurigo nodularis, unspecified itch of the elderly.

The present invention also encompasses processes for producing thecyclic depsipeptide, or derivative thereof, of the invention, forinstance by cultivation of a Chondromyces strain, a variant or a mutantthereof, in a suitable medium, and optionally chemical derivation of theso-produced cyclic depsipeptide, or by expression of the biosynthesisgenes of a Chondromyces strain, a variant or a mutant thereof, in aheterologous microbial host strain, and optionally chemical derivationof the so-produced cyclic depsipeptide.

These processes of the invention can be performed with the strainsChondromyces crocatus (DSM 19329) or Chondromyces robustus (DSM 19330)or Chondromyces apiculatus (DSM 21595).

The present invention hence also relates to isolated Chondromycesmicroorganisms deposited under the accession number DSM 19329 or DSM19330 or DSM 21595 and to cyclic depsipeptides, or derivative thereof,produced by these isolated Chondromyces microorganisms.

The present invention provides:

In embodiment 1, the invention pertains to a cyclic depsipeptide, or aderivative thereof, having the structure of formula (I):

wherein an ester bond is formed between the carboxy group of A7 and thehydroxy group of A2,wherein X and A₁ are each independently optional, and whereinX is H or an amino group modifying moiety and may be typically selectedfrom an aryl carbonyl residue or from an acyl residue,A₁ is glutamine, ornithine, glutamic acid or a derivative thereof;A₂ is threonine or serine,A₃ is leucine,A₄ is Ahp, 3-amino-piperidine-2-one, dehydro-AHP, Ahp-L Ahp-II, proline,5-hydroxy-proline or a derivative thereof, wherein the point of fusion(with A₃ and A₅) are at the nitrogen atom and the carboxyl oxygen (byvirtue of the replacement of a hydrogen atom by a bond) of the proline,and 5-hydroxyproline,wherein Ahp, 3-amino-piperidine-2-one, dehydro-AHP, Ahp-I, and Ahp-II,are as defined below, and wherein the point of fusion (with A₃ and A₅)are at the nitrogen atoms of the said compounds (by virtue of thereplacement of a hydrogen atom by a bond):

wherein X is O or a bond, and R is an organic moiety or is a radical asdefined in embodiment 3,A₅ is isoleucine, phenylalanine or valine,A₆ is tyrosine, N-Me-tyrosine or a derivative thereof,A₇ is leucine, isoleucine or valine,or a pharmaceutically acceptable salt thereof

In embodiment 2, the invention pertains to the depsipeptide ofembodiment 1 wherein X is CH₃CH₂CH(CH₃)CO, (CH₃)₂CHCH₂CO, (CH₃)₂CHCO,CH₃CO or Phenyl-CO.

In embodiment 3, the invention pertains to the depsipeptide ofembodiment 1 wherein the nitrogen atom of the amid bond between A5 andA6 is substituted with a methyl and the OH group of the tyrosine is OR,wherein R is selected from the group consisting of hydrogen,(C₁₋₁₂)alkyl, (C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl,halo(C₂₋₁₂)alkenyl, halo(C₂₋₁₂)alkynyl, (C₁₋₄₂)alkoxycarbonyl,(C₁₋₁₂)alkoxy-carbonyl(C₁₋₁₂)alkyl, (C₁₋₁₂)alkylaminocarbonyl,unsubstituted or further substituted by aryl, arylalkyl, arylalkenyl orarylalkynyl, heterocyclyl and heterocyclylalkyl.

In embodiment 4, the invention pertains to the depsipeptide ofembodiment 1 wherein A4 is the Ahp derivative 3-amino-piperidin-2-one,Ahp-I or Ahp-II, wherein R is selected from the group consisting of(C₁₋₁₂)alkyl, (C₂₋₁₂)al-kenyl, (C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl,(C₁₋₁₂)alkoxy(C₁₋₁₂)alkyl, (C₁₋₁₂)alkoxy(C₁₋₁₂)alkoxy(C₁₋₁₂)alkyl,hydroxy(C₁₋₁₂)alkyl, phenyl and phenyl(C₁₋₆)alkyl.

In embodiment 5, the invention pertains to the depsipeptide ofembodiment 1 wherein the acyl residue X is CH₃CH₂CH(CH₃)CO or(CH₃)₂CHCO,

A₁ is glutamine, glutamic acid, or a derivative thereof,

A₂ is threonine,

A₃ is leucine,

A₄ is Ahp, 3-amino-piperidine-2-one, proline, 5-hydroxy-proline or aderivative thereof,

A₅ is isoleucine,

A₆ is tyrosine, N-Me-tyrosine or a derivative thereof,

A₇ is isoleucine or valine, preferably isoleucine.

In embodiment 6, the invention pertains to the depsipeptide of any ofthe previous embodiments wherein A₄ is Ahp, Ahp-I,3-amino-piperidine-2-one, proline, or 5-hydroxy-proline, preferably Ahp,Ahp-I, 3-amino-piperidine-2-one, or 5-hydroxy-proline, also preferablyAhp, Ahp-I or 5-hydroxy-proline, more preferably Ahp.

In embodiment 7, the invention pertains to a depsipeptide of embodiment1, which is a compound in accordance to formulae A or B,

wherein X and A₁ are as defined in embodiment 1, and whereinR2 the side chain of the amino acid threonine or serine,R3 the side chain of leucine,R5 is the side chain of the amino acid isoleucine or valine, inparticular R5 stands for isoleucine,R6 is the side chain of tyrosine optionally derivatized, in particularoptionally derivatized on its hydroxyl group as defined in embodiment 3,R7 is the side chain of the amino acid leucine, isoleucine or valine, inparticular R7 stands for isoleucine,Y is either hydrogen or a methyl, and Y is in particular methyl.

In embodiment 8, the invention pertains to the cyclic depsipeptide ofany of the previous embodiments wherein A1, A2, A3, A5, A6 and A7 areL-amino acids.

In embodiment 9, the invention pertains to the cyclic depsipeptide ofany of the previous embodiments wherein A4 is 3S,6R Ahp.

In embodiment 10, the invention pertains to the cyclic depsipeptide ofany of the previous embodiments wherein A1 is a glutamine, ornithine, ora glutamine derivative as described in any of the examples of thedescription, and is for example selected from glutamic nitrile, glutamicacid ester such as C₁₋₁₂alkyl ester (e.g. glutamic acid methyl ester) orsuch as C₆₋₂₄aryl ester (e.g. glutamic acid phenyl or benzyl ester).

In embodiment 11, the invention pertains to a pharmaceutical compositioncomprising a cyclic depsipeptide of any of the previous embodiments inconjunction with a pharmaceutical acceptable carrier and/or ingredient.

In embodiment 12, the invention pertains to the cyclic depsipeptide ofany of the previous embodiments for use as a medicament, in particularfor use as described in the methods for treating a patient, such asembodiments 13-15, and the use of the said depsipeptides in themanufacture of a medicament for the treatment in a disease or disorderas described in the said method embodiments.

In embodiments 13, the invention pertains to a method of treating asubject suffering from inflammatory and/or hyperpoliferative andpruritic skin diseases such as keloids, hypertrophic scars, acne, atopicdermatitis, psoriasis, pustular psioriasis, rosacea, Netherton'ssyndrome or other pruritic dermatoses such as prurigo nodularis,unspecified itch of the elderly as well as other diseases withepithelial barrier dysfunction such as aged skin, inflammatory boweldisease and Crohn's disease, as well as pancreatitis, or of cancer, inparticular ovarian cancer, cystic fibrosis (CF), chronic obstructivepulmonary disease (COPD), pulmonary fibrosis, adult respiratory distresssyndrome, chronic bronchitis, hereditary emphysema, rheumatoidarthritis, IBD, psoriasis, asthma, comprising administering to saidsubject a therapeutically effective amount of a cyclic depsipeptide, orderivative thereof, of any of embodiments 1-10.

In embodiment 14, the invention pertains to a method of treating asubject according to embodiment 13, wherein the subject suffers fromkeloids, hypertrophic scars, acne, atopic dermatitis, psoriasis,pustular psioriasis, rosacea, Netherton's syndrome or other pruriticdermatoses such as prurigo nodularis, unspecified itch of the elderly aswell as other diseases with epithelial barrier dysfunction such as agedskin, inflammatory bowel disease and Crohn's disease, as well aspancreatitis, or of cancer, in particular ovarian cancer.

In embodiment 15, the invention pertains to a method of treating asubject according to embodiment 14, wherein the subject suffers fromkeloids, hypertrophic scars, acne, atopic dermatitis, psoriasis,pustular psioriasis, rosacea, Netherton's syndrome or other pruriticdermatoses such as prurigo nodularis, unspecified itch of the elderly aswell as other diseases with epithelial barrier dysfunction such as agedskin.

In embodiment 16, the invention pertains to a method of treating asubject according to embodiment 13, wherein the subject suffers fromcystic fibrosis (CF), chronic obstructive pulmonary disease (COPD),pulmonary fibrosis, adult respiratory distress syndrome, chronicbronchitis, hereditary emphysema, rheumatoid arthritis, IBD, psoriasis,asthma.

In embodiment 17, the invention pertains to a process for producing thecyclic depsipeptide, or derivative thereof, of any of embodiments 1-10comprising cultivation of a Chondromyces strain, a variant or a mutantthereof, in a suitable medium, and optionally chemical derivation of theso-produced cyclic depsipeptide.

In embodiment 18, the invention pertains to a process for producing thecyclic depsipeptide, or derivative thereof, of any of embodiments 1-10comprising expressing the biosynthesis genes of a Chondromyces strain, avariant or a mutant thereof, in a heterologous microbial host strain,and optionally chemical derivation of the so-produced cyclicdepsipeptide.

In embodiment 19, the invention pertains to the process of embodiment 17or 18 wherein the strain is Chondromyces crocatus (DSM 19329) orChondromyces robustus (DSM 19330) or Chondromyces apiculatus (DSM21595).

In embodiment 20, the invention pertains to an isolated Chondromycesmicroorganism producing the cyclic depsipeptide, or derivative thereof,of any of embodiments 1-10, deposited under the accession number DSM19329 or DSM 19330 or DSM 21595.

In embodiment 21, the invention pertains to a cyclic depsipeptide, orderivative thereof, produced by the isolated Chondromyces microorganismof embodiment 20 or obtained by a process according to claims 17-18.

In embodiment 22, the invention pertains to a process for thepreparation of a derivative of a cyclic depsipeptide, or derivativethereof, according to embodiment 1 which comprises alternatively

a)—the preparation of a derivative of a cyclic depsipeptide, orderivative thereof, according to embodiment 1 wherein A4 is

by treatment of a compound wherein A4 is

with an organic or inorganic acid, or a Lewis acid at a temperaturebetween −78° C. and 150° C.;b)—the preparation of a derivative of a cyclic depsipeptide, orderivative thereof, according to embodiment 1 wherein A4 is

by treatment of a compound wherein A4 is

with molecular hydrogen or source thereof in presence of a catalyst in asolvent at a temperature between −50 and 100° C.;c)—the preparation of a derivative of a cyclic depsipeptide, orderivative thereof, according to embodiment 1 wherein A4 is

by treatment of a compound wherein A4 is

with an organic or inorganic acid or a Lewis acid, in presence of anreducing agent at a temperature between −78° C. and 150° C.; ord)—the preparation of a derivative of a cyclic depsipeptide, orderivative thereof, according to embodiment 1 wherein A4 is

by treatment of a compound wherein A4 is

with a substituted or unsubstituted alkanol and an organic or inorganicacid, or a Lewis acid, at a temperature between −78° C. and 150° C.;e)—the preparation of compounds wherein A1 is

wherein n=1,2 and A4 is

wherein R preferentially is H, alkyl, substituted alkyl, by treatment ofa compound wherein A1 is Gln or Asn and A4 is

wherein R preferably is H, alkyl, substituted alkyl, with an substitutedor unsubstituted alkanol and an organic or inorganic acid or a Lewisacid in a solvent or without a solvent at a temperature between −78° C.and 150° C.;f)—the preparation of compounds wherein A1 is

wherein R preferably is H, OH, O-alkyl, substituted O-alkyl, O-acyl, bytreatment of a compound wherein A1 is Gln or Asn and A4 is

with an dehydrating agent in a solvent or without a solvent at atemperature between −78° C. and 150° C.;g)—the preparation of compounds wherein A4 is

and A6 is

wherein R preferably is alkyl, substituted alkyl, acyl, alkoxycarbonylby treatment of a compound wherein A4 is

and A6 is Tyr, with an alkylating agent or an acylating agent in asolvent or without a solvent at a temperature between −78° C. and 150°C.

In embodiment 23, the invention pertains to a cyclic depsipeptide, or aderivative thereof, or a pharmaceutically acceptable salt thereof, inparticular and essentially as described in the description and/or theworking examples.

Specific embodiments of cyclic depsipeptides of the invention are:

The cyclic depsipeptides of formula (II)-(VII), (XI)-(XIV) and (XVII)can be produced by the Chondromyces crocatus strain of the invention(DSM 19329).

Other specific embodiments of cyclic depsipeptides of the invention are:

The cyclic depsipeptides of formula (VIII)-(X) can be produced by theChondromyces robustus of the invention (DSM 19330).

Other specific embodiments of cyclic depsipeptides of the invention are:

The cyclic depsipeptides of formula (XV)-(XVI) can be produced by theChondromyces apculatus of the invention (DSM 21595).

LIST OF ABBREVIATIONS

Ahp 3-amino-6-hydroxy-2-piperidone

DSMZ Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH

hK7 Human kallikrein 7

HPLC High performance/pressure liquid chromatography

HTS High Throughput Screening

IC Intermediary culture

ID Identification

MB Myxobacteria

MC Main-culture

PC Pre-culture

pO₂ Partial pressure of oxygen in culture broth (100%=saturation withair)

rpm Rotations per minute

SCCE Stratum corneum chymotryptic enzyme

SPEX Solid phase extraction

vvm Aeration rate (Volume of air per culture volume and per minute)

A “chemical residue” can be any organic or anorganic chemical moiety.The expression “chemical residue” includes, but is not limited tosubstituted or unsubstituted aliphatic group, e.g. C₁-C₃ alkyl, C₁-C₆alkyl, or C₁-C₁₂ alkyl, substituted or unsubstituted aryl, substitutedor unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, orhalogen. For instance, a chemical residue as defined in the claims canbe any of the chemical groups described herein-below.

The expression “chemical residue” includes, but is not limited to aminoacids, peptides, polypeptides, proteins and the like.

Examples of anorganic chemical moiety are for instance halogens, such asBr or Cl.

An “aliphatic group” is non-aromatic moiety that may contain anycombination of carbon atoms, hydrogen atoms, halogen atoms, oxygen,nitrogen or other atoms, and optionally contain one or more units ofunsaturation, e.g., double and/or triple bonds. An aliphatic group maybe straight chained, branched or cyclic and preferably contains betweenabout 1 and about 24 carbon atoms, more typically between about 1 andabout 12 carbon atoms. In addition to aliphatic hydrocarbon groups,aliphatic groups include, for example, polyalkoxyalkyls, such aspolyalkylene glycols, polyamines, and polyimines, for example. Suchaliphatic groups may be further substituted.

The terms “C₁-C₃ alkyl”, “C₁-C₆ alkyl,” or “C₁-C₁₂ alkyl,” as usedherein, refer to saturated, straight- or branched-chain hydrocarbonradicals containing between one and three, one and twelve, or one andsix carbon atoms, respectively. Examples of C₁-C₃ alkyl radicals includemethyl, ethyl, propyl and isopropyl radicals; examples of C₁-C₆ alkylradicals include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, ted-butyl, sec-butyl, n-pentyl, neopentyl andn-hexyl radicals; and examples of C₁-C₁₂ alkyl radicals include, but arenot limited to, ethyl, propyl, isopropyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl radicals and the like.

The term “substituted alkyl,” as used herein, refers to an alkyl, suchas a C₁-C₁₂ alkyl or C₁-C₆ alkyl group, substituted by one, two, threeor more aliphatic substituents.

Suitable aliphatic substituents include, but are not limited to, —F,—Cl, —Br, —I, —OH, protected hydroxy, aliphatic ethers, aromatic ethers,oxo, —NO₂, —CN, —C₁-C₁₂-alkyl optionally substituted with halogen (suchas perhaloalkyls), C₂-C₁₂-alkenyl optionally substituted with halogen,—C₂-C₁₂-alkynyl optionally substituted with halogen, —NH₂, protectedamino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkenyl,—NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocycloalkyl,-dialkylamino, -diarylamino, -diheteroarylamino, —O—C₁-C₁₂-alkyl,—O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkynyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl,—O-hetero aryl, —O-heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl,—C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkynyl, —C(O)—C₃-C₁₂-cycloalkyl,—C(O)-aryl, —C(O)-heteroaryl, —C(O)-heterocycl-C₁₂-alkyl,—CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkynyl, —CONH—C₃-C₁₂-cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl, —CO₂—C₁-C₁₂-alkyl,—CO₂—C₂-C₁₂-alkenyl, —CO₂—C₂-C₁₂-alkynyl, —CO₂—C₃-C₁₂-cycloalkyl,—CO₂-aryl, —CO₂-heteroaryl, —CO₂-hetero cycloalkyl, —OCO₂—C₁-C₁₂-alkyl,—OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkynyl, —OCO₂—C₃-C₁₂-cycloalkyl,—OCO₂-aryl, —OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂,—OCONH—C₁-C₁₂-alkyl, —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkynyl,—OCONH—C₃-C₁₂-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl,—OCONH-heterocycloalkyl, —NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl,—NHC(O)—C₂-C₁₂-alkynyl, —NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl,—NHC(O)-heteroaryl, —NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl,—NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₂-C₁₂-alkynyl, —NHCO₂—C₃-C₁₂-cycloalkyl,—NHCO₂-aryl, —NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkynyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NR₂,NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkynyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NR-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkynyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkynyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NR)NH—C₁-C₁₂-alkyl, —C(NH)NH—C₂-C₁₂—alkenyl, —C(NR)NH—C₂-C₁₂-alkynyl, —C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH—aryl, —C(NH)NH-heteroaryl, —C(NH)NH-heterocycloalkyl,—S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl, —S(O)—C₂-C₁₂-alkynyl,—S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl, —S(O)-heteroaryl,—S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkynyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkynyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl,-heteroaryl, -hetero arylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkynyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocyclo alkyl, ormethylthiomethyl. It is understood that the aryls, heteroaryls, alkylsand the like can be further substituted.

The terms “C₂-C₁₂ alkenyl” or “C₂-C₆ alkenyl,” as used herein, denote amonovalent group derived from a hydrocarbon moiety containing from twoto twelve or two to six carbon atoms having at least one carbon-carbondouble bond by the removal of a single hydrogen atom. Alkenyl groupsinclude, but are not limited to, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, alkadienes and the like.

The term “substituted alkenyl,” as used herein, refers to a “C₂-C₁₂alkenyl” or “C₂-C₆ alkenyl” group as previously defined, substituted byone, two, three or more aliphatic substituents.

The terms “C₂-C₁₂ alkynyl” or “C₂-C₆ alkynyl,” as used herein, denote amonovalent group derived from a hydrocarbon moiety containing from twoto twelve or two to six carbon atoms having at least one carbon-carbontriple bond by the removal of a single hydrogen atom. Representativealkynyl groups include, but are not limited to, for example, ethynyl,1-propynyl, 1-butynyl, and the like.

The term “substituted alkynyl,” as used herein, refers to a “C₂-C₁₂alkynyl” or “C₂-C₆ alkynyl” group as previously defined, substituted byone, two, three or more aliphatic substituents.

The term “C₁-C₆ alkoxy,” as used herein, refers to a C₁-C₆ alkyl group,as previously defined, attached to the parent molecular moiety throughan oxygen atom. Examples of C₁-C₆-alkoxy include, but are not limitedto, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy and n-hexoxy.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The term “aryl,” as used herein, refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyland the like.

The term “substituted aryl,” as used herein, refers to an aryl group, aspreviously defined, substituted by one, two, three or more aromaticsubstituents.

Aromatic substituents include, but are not limited to, —F, —Cl, —Br, —I,—OH, protected hydroxy, aliphatic ethers, aromatic ethers, oxo, —NO₂,—CN, —C₁-C₁₂-alkyl optionally substituted with halogen (such asperhaloalkyls), C₂-C₁₂-alkenyl optionally substituted with halogen,—C₂-C₁₂-alkynyl optionally substituted with halogen, —NH₂, protectedamino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkenyl,—NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocycloalkyl,-dialkylamino, -diarylamino, -diheteroarylamino, —O—C₁-C₁₂-alkyl,—O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkynyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl,—O-hetero aryl, —O-heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl,—C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkynyl, —C(O)—C₃-C₁₂-cycloalkyl,—C(O)-aryl, —C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH₂,—CONH—C₁-C₁₂-alkyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkynyl,—CONH—C₃-C₁₂-cycloalkyl, —CONH-aryl, —CONH-heteroaryl,—CONH-heterocycloalkyl, —CO₂—C₁-C₁₂-alkyl, —CO₂—C₂-C₁₂-alkenyl,—CO₂—C₂-C₁₂-alkynyl, —CO₂—C₃-C₁₂-cycloalkyl, —CO₂-aryl, —CO₂-heteroaryl,—CO₂-hetero cycloalkyl, —OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl,—OCO₂—C₂-C₁₂-alkynyl, —OCO₂—C₃-C₂-cycloallyl, —OCO₂-aryl,—OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkynyl, —OCONH—C₃-C₁₂-cycloalkyl,—OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl, —NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkynyl,—NHC(O)—C₃-C₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkynyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkynyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkynyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkynyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkynyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(N_(H))-heteroaryl,—NHC(N_(H))-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkynyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkynyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₂-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkynyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkynyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂ CH₃, -aryl, -arylalkyl,-heteroaryl, -hetero arylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C_(12-alkyl, —S—C) ₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkynyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl. It is understood that the aryls, heteroaryls, alkylsand the like can be further substituted.

The term “arylalkyl,” as used herein, refers to an aryl group attachedto the parent compound via a C₁-C₃ alkyl or C₁-C₆ alkyl residue.Examples include, but are not limited to, benzyl, phenethyl and thelike.

The term “substituted arylalkyl,” as used herein, refers to an arylalkylgroup, as previously defined, substituted by one, two, three or morearomatic substituents.

The term “heteroaryl,” as used herein, refers to a mono-, bi-, ortri-cyclic aromatic radical or ring having from five to ten ring atomsof which at least one ring atom is selected from S, O and N; zero, oneor two ring atoms are additional heteroatoms independently selected fromS, O and N; and the remaining ring atoms are carbon, wherein any N or Scontained within the ring may be optionally oxidized. Heteroarylincludes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. The heteroaromatic ring may be bonded to the chemical structurethrough a carbon or hetero atom.

The term “substituted heteroaryl,” as used herein, refers to aheteroaryl group as previously defined, substituted by one, two, threeor four aromatic substituents.

The term “C₃-C₁₂-cycloalkyl,” as used herein, denotes a monovalent groupderived from a monocyclic or bicyclic saturated carbocyclic ringcompound by the removal of a single hydrogen atom. Examples include, butnot limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl.

The term “substituted C₃-C₁₂-cycloalkyl,” as used herein, refers to aC₃-C₁₂-cycloalkyl group as previously defined, substituted by one, two,three or more aliphatic substituents.

The term “heterocycloalkyl,” as used herein, refers to a non-aromatic5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused system,where (i) each ring contains between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, (ii) each5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms mayoptionally be oxidized, (iv) the nitrogen heteroatom may optionally bequaternized, (iv) any of the above rings may be fused to a benzene ring,and (v) the remaining ring atoms are carbon atoms which may beoptionally oxo-substituted. Representative heterocycloalkyl groupsinclude, but are not limited to, [1,3]dioxolane, pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, and tetrahydrofuryl.

The term “substituted heterocycloalkyl,” as used herein, refers to aheterocycloalkyl group, as previously defined, substituted by one, two,three or more aliphatic substituents. The term “heteroarylalkyl,” asused herein, to an heteroaryl group attached to the parent compound viaa C₁-C₃ alkyl or C₁-C₆ alkyl residue. Examples include, but are notlimited to, pyridinylmethyl, pyrimidinylethyl and the like.

The term “substituted heteroarylalkyl,” as used herein, refers to aheteroarylalkyl group, as previously defined, substituted by independentreplacement of one, two, or three or more aromatic substituents.

The term “C₁-C₃-alkylamino,” as used herein, refers to one or twoC₁-C₃-alkyl groups, as previously defined, attached to the parentmolecular moiety through a nitrogen atom. Examples of C₁-C₃-alkylaminoinclude, but are not limited to, methylamino, dimethylamino, ethylamino,diethylamino, and propylamino.

The term “alkylamino” refers to a group having the structure —NH(C₁-C₁₂alkyl) where C₁-C₁₂ alkyl is as previously defined.

The term “dialkylamino” refers to a group having the structure —N(C₁-C₁₂alkyl) (C₁-C₁₂ alkyl), where C₁-C₁₂ alkyl is as previously defined.Examples of dialkylamino are, but not limited to, dimethylamino,diethylamino, methylethylamino, piperidino, and the like.

The term “alkoxycarbonyl” represents an ester group, i.e., an alkoxygroup, attached to the parent molecular moiety through a carbonyl groupsuch as methoxycarbonyl, ethoxycarbonyl, and the like.

The term “carboxaldehyde,” as used herein, refers to a group of formula—CHO.

The term “carboxy,” as used herein, refers to a group of formula —COOH.

The term “carboxamide,” as used herein, refers to a group of formula—C(O)NH(C₁-C₁₂ alkyl) or —C(O)N(C₁-C₁₂ alkyl) (C₁-C₁₂alkyl), —C(O)NH₂,NHC(O)(C₁-C₁₂ alkyl), N(C₁-C₁₂alkyl)C(O)(C₁-C₁₂ alkyl) and the like.

The term “hydroxy protecting group” as used herein, refers to a labilechemical moiety which is known in the art to protect a hydroxyl groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the hydroxy protecting group as described hereinmay be selectively removed. Hydroxy protecting groups as known in theare described generally in T. H. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York(1999). Examples of hydroxyl protecting groups includebenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl,isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl,trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl,2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, 1,1-dimethyl-2-propenyl,3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl,triphenylmethyl (trityl), tetrahydrofuryl, methoxymethyl,methylthiomethyl, benzyloxymethyl, 2,2,2-triehloroethoxymethyl,2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like.Preferred hydroxyl protecting groups for the present invention areacetyl (Ac or —C(O)CH₃), benzoyl (Bn or —C(O)C₆H₅), and trimethylsilyl(TMS or —Si(CH₃)₃).

The term “protected hydroxy”, as used herein, refers to a hydroxy groupprotected with a hydroxy protecting group, as defined above, includingbenzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl groups,for example.

The term “amino protecting group”, as used herein, refers to a labilechemical moiety which is known in the art to protect an amino groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the amino protecting group as described hereinmay be selectively removed. Amino protecting groups as known in the aredescribed generally in T. H. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).Examples of amino protecting groups include, but are not limited to,t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and thelike.

The term “protected amino”, as used herein, refers to an amino groupprotected with an amino protecting group as defined above.

The term “acyl” includes residues derived from acids, including but notlimited to carboxylic acids, carbamic acids, carbonic acids, sulfonicacids, and phosphorous acids. Examples include aliphatic carbonyls,aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphaticsulfinyls, aromatic sulfonyls, aliphatic sulfamyls, aromatic sulfamyls,aromatic phosphates and aliphatic phosphates.

An “amino acid” is a molecule that contains both amine and carboxylfunctional groups with the general formula NH2CHRCOOH. The term aminoacid includes standard amino acids and nonstandard amino acids.

“Standard amino acids” are alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

A “standard amino acid which is not aspartic acid” is selected from thegroup consisting of alanine, arginine, asparagine, cysteine, glutamicacid, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine. In the case of glutamine or glutamic acid, a“derivative thereof” is e.g. a nitrile or an ester, such as e.g.glutamine-nitrile, glutamic acid ester.

“Nonstandard amino acids” are amino acids (molecules that contains bothamine and carboxyl functional groups) which are not one of the standardamino acids. Examples thereof are selenocysteine (incorporated into someproteins at a UGA codon), pyrrolysine (used by some methanogenicbacteria in enzymes to produce methane and coded for with the codonUAG), lanthionine, 2-aminoisobutyric acid, dehydroalanine,3-amino-6-hydroxy-2-piperidone, gamma-aminobutyric acid, ornithine,citrulline, homocysteine, dopamine or hydroxyproline.

“Non-basic standard amino acids” are alanine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine.

“Ahp” (3-amino-6-hydroxy-piperidin-2-one) is a nonstandard amino acidfound for instance in cyanobacteria. “Ahp derivatives” include, but arenot limited to 3-amino-3,4-dihydro-1H-pyridin-2-one (dehydro-AHP),3-amino-piperidin-2-one and “ether and ester derivatives of AHP.Preferred Ahp derivatives are 3-amino-piperidin-2-one, or Ahp-I orAhp-II as depicted below wherein R is selected from the group consistingof (C₁₋₁₂)alkyl, (C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl halo(C₁₋₁₂)alkyl,(C₁₋₁₂)alkoxy(C₁₋₁₂)alkyl, (C₁₋₁₂)alkoxy(C₁₋₁₂)alkoxy(C₁₋₁₂)alkyl,hydroxy(C₁₋₁₂)alkyl, phenyl and phenyl(C₁₋₆)alkyl.

Different members of this family of nonstandard amino acids are:

Proline derivative includes e.g. 5-hydroxyprolin.

“Amino acid derivatives” include, but are not limited to, O-alkyl,O-aryl, O-acyl, S-alkyl, S-aryl, S—S-alkyl, alkoxycarbonyl,O-carbonyl-alkoxy, carbonate, O-carbonyl-aryloxy, O-carbonyl-alkylamino,O-carbonyl-arylamino, N-alkyl, N-dialkyl, N-trialkylammonium, N-acyl,N-carbonyl-alkoxy, N-carbonyl-aryloxy, N-carbonyl-alkylamino,N-carbonyl-arylamino, N-sulfonylalkyl, or N-sulfonylaryl.

“Non-basic standard amino acid derivatives” include, but are not limitedto, O-alkyl, O-aryl, O-acyl, S-alkyl, S-aryl, S—S-alkyl, alkoxycarbonyl,O-carbonyl-alkoxy, carbonate, O-carbonyl-aryloxy, O-carbonyl-alkylamino,O-carbonyl-arylamino, N-alkyl, N-dialkyl, N-trialkylammonium, N-acyl,N-carbonyl-alkoxy, N-carbonyl-aryloxy, N-carbonyl-alkylamino,N-carbonyl-arylamino, N-sulfonylalkyl, or N-sulfonylaryl.

“Tyrosine derivative” include, but are not limited to, —O-alkyl, O-aryl,O-heteroaryl, O-acyl, O—PO₃H and O—SO₃H, as well as halogenation, inortho or meta position.

The OH group of the tyrosine may be OR, wherein R is selected from thegroup consisting of

hydrogen, (C₁₋₁₂)alkyl, (C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl,halo(C₁₋₁₂)alkyl,

halo(C₂₋₁₂)alkenyl, halo(C₂₋₁₂)alkynyl, (C₁₋₁₂)alkoxycarbonyl,

(C₁₋₁₂)alkoxycarbonyl(C₁₋₁₂)alkyl,

(C₁₋₁₂)alkylaminocarbonyl, unsubstituted or further substituted by aryl,arylalkyl, arylalkenyl or arylalkynyl, heterocyclyl andheterocyclylalkyl.

“Depsipeptide derivative” include but are not limited to, depsipeptidesmodified as described herein and to those specifically described in theexamples below. Said derivatives can be prepared using methods wellknown in the art.

The invention further relates to pharmaceutically acceptable salts andderivatives of the compounds of the present invention and to methods forobtaining such compounds. One method of obtaining the compound is bycultivating a Chondromyces strain of the invention, or a mutant or avariant thereof, under suitable conditions, preferably using thefermentation protocol described herein-below.

“Salts” of compounds of the present invention having at least onesalt-forming group may be prepared in a manner known per se. Forexample, salts of compounds of the present invention having acid groupsmay be formed, for example, by treating the compounds with metalcompounds, such as alkali metal salts of suitable organic carboxylicacids, e.g., the sodium salt of 2-ethylhexanoic acid, with organicalkali metal or alkaline earth metal compounds, such as thecorresponding hydroxides, carbonates or hydrogen carbonates, such assodium or potassium hydroxide, carbonate or hydrogen carbonate, withcorresponding calcium compounds or with ammonia or a suitable organicamine, stoichiometric amounts or only a small excess of the salt-formingagent preferably being used. Acid addition salts of compounds of thepresent invention are obtained in customary manner, e.g., by treatingthe compounds with an acid or a suitable anion exchange reagent.Internal salts of compounds of the present invention containing acid andbasic salt-forming groups, e.g., a free carboxy group and a free aminogroup, may be formed, e.g., by the neutralisation of salts, such as acidaddition salts, to the isoelectric point, e.g., with weak bases, or bytreatment with ion exchangers.

Salts can be converted in customary manner into the free compounds;metal and ammonium salts can be converted, for example, by treatmentwith suitable acids, and acid addition salts, for example, by treatmentwith a suitable basic agent.

Mixtures of isomers obtainable according to the invention can beseparated in a manner known per se into the individual isomers;diastereoisomers can be separated, for example, by partitioning betweenpolyphasic solvent mixtures, recrystallisation and/or chromatographicseparation, for example over silica gel or by, e.g., medium pressureliquid chromatography over a reversed phase column, and racemates can beseparated, for example, by the formation of salts with optically puresalt-forming reagents and separation of the mixture of diastereoisomersso obtainable, for example by means of fractional crystallisation, or bychromatography over optically active column materials.

Intermediates and final products can be worked up and/or purifiedaccording to standard methods, e.g., using chromatographic methods,distribution methods, (re-) crystallization, and the like.

The cyclic depsipeptides of the invention can inhibit ofchymotrypsin-like proteases. Examples of chymotrypsin-like proteases areelastases and kallikrein 7. In particular, the cyclic depsipeptides ofthe invention are excellent inhibitors of kallikrein 7.

An “inhibitor” is a cyclic depsipeptide that inhibits an enzymaticreaction with a measure IC₅₀ of less than 100 μM, for instance 50 μM, 30μM, 20 μM or 10 μM. Particularly preferred are cyclic depsipeptides withan IC₅₀ of less than 30 μM for human kallikrein 7, for instance cyclicdepsipeptides with an IC₅₀ of less than 10 μM, 1 μM, 100 nM, 50 nM, 40nM, 30 nM, 20 nM, 10 nM, or less. E.g. compounds of examples 5, 19 and33 show IC50 values of 0.009 μM, 0.007 μM, 0.005 μM, respectively. IC₅₀for human kallikrein can be measured using the fluorescence-quenchedsubstrateAc-Glu-Asp(EDANS)-Lys-Pro-Ale-Leu-Phe^Arg-Leu-Gly-Lys(DABCYL)-Glu-NH₂(where ^ indicates the scissile bond, identified by MS analysis) whichcan be purchased from Biosyntan (Berlin, Germany). Enzymatic reactionsare conducted in 50 mM sodium citrate buffer at pH 5.6 containing 150 mMNaCl and 0.05% (w/v) CHAPS. For the determination of IC₅₀ values theassay is performed at room temperature in 384-well plates. All finalassay volumes are 30 μl. Test compounds are dissolved in 90% (v/v)DMSO/water and diluted in water (containing 0.05% (w/v) CHAPS) to3-times the desired assay concentration. The 11 final compoundconcentrations are: 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1μM, 3 μM, 10 μM and 30 μM. For each assay, 10 μl water/CHAPS (±testcompound) are added per well, followed by 10 μl protease solution(diluted with 1.5× assay buffer). The protease concentration in finalassay solution is 0.2 nM (according to the enzyme concentrationsdetermined by the Bradford method). After 1 hour of incubation at roomtemperature, the reaction is started by addition of 10 μl substratesolution (substrate dissolved in 1.5× assay buffer, final concentrationis 2 μM). The effect of the compound on the enzymatic activity isobtained from the linear progress curves and determined from tworeadings, the first one taken directly after the addition of substrate(t=0 min) and the second one after 1 hour (t=60 min). The IC₅₀ value iscalculated from the plot of percentage of inhibition vs. inhibitorconcentration using non-linear regression analysis software (XLfit,Vers. 4.0; ID Business Solution Ltd., Guildford, Surrey, UK).

“Diseases” and “disorders” which may hence be treated or prevented usingthe cyclic depsipeptides, are diseases known to be related withchymotrypsin-like proteases. More preferred are diseases known to berelated to elastases or kallikrein 7 activity. Equally preferred arediseases known to be related to human neutrophil elastase. Diseases anddisorders which may hence be treated or prevented using the cyclicdepsipeptides of the invention include pain, acute inflammation, chronicinflammation, arthritis, inflamed joints, bursitis, osteoarthritis,rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis,fibromyalgia, systemic lupus erythematosus, phlebitis, tendinitis, rash,psoriasis, acne, eczema, facial seborrheic eczema, eczema of the hands,face or neck, foreskin infections, athlete's foot, fistulae infections,infected topical ulcers, navel infections in newborns, wrinkles, scars,kelloids, boils, warts and allergic itch, hemorrhoids, wounds, woundinfections, wounds from burns, a fungal infection and an immunogicaldisorder including an autoimmune disease. Preferred diseases which maybe treated or prevented using the cyclic depsipeptides of the inventioninclude chronic obstructive pulmonary disease (including pulmonaryemphysema and chronic bronchitis), chronic and acute interstitialpneumonia, idiopathic interstitial pneumonia (IIP), diffusepanbronchiolitis, cystic lung fibrosis, acute lung injury (ALI)/acuterespiratory distress syndrome (ARDS), bronchiectasis, asthma,pancreatitis, nephritis, hepatitis (hepatic failure), chronic rheumatoidarthritis, arthrosclerosis, osteroarthritis, psoriasis, periodontaldisease, atherosclerosis, organ transplant rejection, tissue injurycaused by ischemic/reperfusion, shock, septicemia, blood coagulopathyincluding disseminated intravascular coagulation (DIC) and deepvein-thrombosis, conjunctivitis, keratitis, corneal ulcer, Crohn'sdisease, systemic lupus erythematosus. More preferred diseases anddisorders which may be treated or prevented using the cyclicdepsipeptides of the invention are “epithelial dysfunction” or“epithelial disease” including, but are not limited to, inflammatoryand/or hyperpoliferative and pruritic skin diseases such as keloids,hypertrophic scars, acne, atopic dermatitis, psoriasis, Netherton'ssyndrome or other pruritic dermatoses such as prurigo nodularis,unspecified itch of the elderly as well as other diseases withepithelial barrier dysfunction such as inflammatory bowel disease andCrohn's disease, cystic fibrosis (CF), chronic obstructive pulmonarydisease (COPD), pulmonary fibrosis, adult respiratory distress syndrome,chronic bronchitis, hereditary emphysema, rheumatoid arthritis, IBD,psoriasis, asthma. In another preferred embodiment, the cyclicdepsipeptides of the invention can be use to treat cancer, in particularovarian cancer.

Diseases and disorders which may hence be preferably treated orprevented using the cyclic depsipeptides of the invention includeinflammatory and/or hyperpoliferative and pruritic skin diseases such askeloids, hypertrophic scars, acne, atopic dermatitis, psoriasis,pustular psioriasis, rosacea, Netherton's syndrome or other pruriticdermatoses such as prurigo nodularis, unspecified itch of the elderly aswell as other diseases with epithelial barrier dysfunction such as agedskin, inflammatory bowel disease and Crohn's disease, as well aspancreatitis, or of cancer, in particular ovarian cancer, cysticfibrosis (CF), chronic obstructive pulmonary disease (COPD), pulmonaryfibrosis, adult respiratory distress syndrome, chronic bronchitis,hereditary emphysema, rheumatoid arthritis, IBD, psoriasis, and asthma.

Diseases and disorders which may hence be more preferably treated orprevented using the cyclic depsipeptides of the invention includekeloids, hypertrophic scars, acne, atopic dermatitis, psoriasis,pustular psioriasis, rosacea, Netherton's syndrome or other pruriticdermatoses such as prurigo nodularis, unspecified itch of the elderly aswell as other diseases with epithelial barrier dysfunction such as agedskin, inflammatory bowel disease and Crohn's disease, as well aspancreatitis, or of cancer, in particular ovarian cancer.

Diseases and disorders which may hence be equally preferably treated orprevented using the cyclic depsipeptides of the invention include cysticfibrosis (CF), chronic obstructive pulmonary disease (COPD), pulmonaryfibrosis, adult respiratory distress syndrome, chronic bronchitis,hereditary emphysema, rheumatoid arthritis, IBD, psoriasis, asthma.

Diseases and disorders which may be even more preferably treated orprevented using the cyclic depsipeptides of the invention includekeloids, hypertrophic scars, acne, atopic dermatitis, psoriasis,pustular psioriasis, rosacea, Netherton's syndrome or other pruriticdermatoses such as prurigo nodularis, unspecified itch of the elderly,as well as other diseases with epithelial barrier dysfunction such asaged skin.

Human kallikrein 7 (hK7) is an enzyme with serine protease activitylocated in the human skin. It was first described as stratum corneumchymotryptic enzyme (SCCE) and may play a role in desquamation ofstratum corneum by cleaving proteins of the stratum corneum (e.g.,corneodesmosin and plakoglobin). The stratum corneum is thebarrier-forming outermost layer of the epidermis and consists ofcornified epithelial cells surrounded by highly organized lipids. It iscontinuously being formed by epidermal differentiation and in normalepidermis the constant thickness of the stratum corneum is maintained bya balance between the proliferation of the keratinocytes anddesquamation. Enhanced expression of SCCE in inflammatory skin diseasemay be of etiological significance (Hansson, et al. (2002)). Transgenicmice expressing human kallikrein 7 in epidermal keratinocytes were foundto develop pathologic skin changes with increased epidermal thickness,hyperkeratosis, dermal inflammation, and severe pruritis. A geneticassociation between a 4 bp (AACC) insertion in the 3′UTR of the stratumcorneum chymotryptic enzyme gene and atopic dermatitis has been reported(Vasilopoulos, et al. (2004)), suggesting that the enzyme could have animportant role in the development of atopic dermatitis. Atopicdermatitis is a disease with an impaired skin barrier that affects15%-20% of children.

Kallikrein 7 is a S1 serine protease of the kallikrein gene familydisplaying a chymotrypsin like activity. Human kallikrein 7 (hK7, KLK7or stratum corneum chymotryptic enzyme (SCCE), Swissprot P49862) playsan important role in skin physiology (1, 2, 3). It is mainly expressedin the skin and has been reported to play an important role in skinphysiology. hK7 is involved in the degradation of the intercellularcohesive structures in cornified squamous epithelia in the process ofdesquamation. The desquamation process is well regulated and delicatelybalanced with the de 120170 production of corneocytes to maintain aconstant thickness of the stratum corneum, the outermost layer of theskin critically involved in skin barrier function. In this regard, hK7is reported to be able to cleave the corneodesmosomal proteinscorneodesmosin and desmocollin 1 (4, 5, 6). The degradation of bothcorneodesmosomes is required for desquamation. In addition, veryrecently it has been shown that the two lipid processing enzymesβ-glucocerebrosidase and acidic sphingomyelinase can be degraded by hK7(7). Both lipid processing enzymes are co-secreted with their substratesglucosylceramides and sphingomyelin and process these polar lipidprecursors into their more non-polar products e.g. ceramides, which aresubsequently incorporated into the extracellular lamellar membranes. Thelamellar membrane architecture is critical for a functional skinbarrier. Finally, hK7 has been shown to activate Interleukin-1β (IL-1β)precursor to its active form in vitro (8). Since keratinocytes expressIL-1β but not the active form of the specific IL-1β converting enzyme(ICE or caspase 1), it is proposed that IL-1β activation in humanepidermis occurs via another protease, a potential candidate being hK7.

Recent studies link an increased activity of hK7 to inflammatory skindiseases like atopic dermatitis, psoriasis or Netherton's syndrome. Thismight lead to an uncontrolled degradation of corneodesmosomes resultingin a miss-regulated desquamation, an enhanced degradation of lipidprocessing enzymes resulting in a disturbed lamellar membranearchitecture or an uncontrolled activation of the proinflammatorycytokine IL-1β. The net result would be an impaired skin barrierfunction and inflammation (see also WO-A-20041108139).

Due to the fact that the hK7 activity is controlled at several levels,various factors might be responsible for an increased hK7 activity ininflammatory skin diseases. Firstly, the amount of protease beingexpressed might be influenced by genetic factors. Such a genetic link, apolymorphism in the 3′-UTR in the hK7 gene, was recently described (9).The authors hypothesise that the described 4 base pair insertion in the3′-UTR of the kallikrein 7 gene stabilizes the hK7 mRNA and results inan overexpression of hK7. Secondly, since hK7 is secreted via lamellarbodies to the stratum corneum extracellular space as zymogen and it isnot able to autoactivate, it needs to be activated by another proteasee.g. hK5 (5). Uncontrolled activity of such an activating enzyme mightresult in an overactivation of hK7. Thirdly, activated hK7 can beinhibited by natural inhibitors like LEKTI, ALP or elafin (10, 11). Thedecreased expression or the lack of such inhibitors might result in anenhanced activity of hK7. Recently it was found, that mutations in thespink5 gene, coding for LEKTI, are causative for Netherton's syndrome(12) and a single point mutation in the gene is linked to atopicdermatitis (13, 14). Finally, another level of controlling the activityof hK7 is the pH. hK7 has a neutral to slightly alkaline pH optimum (2)and there is a pH gradient from neutral to acidic from the innermost tothe outermost layers in the skin. Environmental factors like soap mightresult in a pH increase in the outermost layers of the stratum corneumtowards the pH optimum of hK7 thereby increasing the hK7 activity.

An increased activity of hK7 is linked to skin diseases with an impairedskin barrier including inflammatory and hyperpoliferative skin diseases.Firstly, Netherton's syndrome patients show a phenotype dependentincrease in serine protease activity, a decrease in corneodesmosomes, adecrease in the lipid processing enzymes β-glucocerebrosidase and acidicsphingomyelinase, and an impaired barrier function (15, 16). Secondly, atransgenic mice overexpressing human kallikrein 7 shows a skin phenotypesimilar to that found in patients with atopic dermatitis (17, 18, 19).Thirdly, in the skin of atopic dermatitis and psoriasis patientselevated levels of hK7 were described (17, 20). Furthermore, increasedactivity of K7 and thus epithelial barrier dysfunction may also play animportant role in the pathology of other epithelial diseases such asinflammatory bowel disease and Crohn's disease.

Therefore, hK7 is considered to be a potential target for the treatmentof diseases involved with epithelial dysfunction such as inflammatoryand/or hyperpoliferative and pruritic skin diseases such as keloids,hypertrophic scars, acne, atopic dermatitis, psoriasis, pustularpsioriasis, rosacea, Netherton's syndrome or other pruritic dermatosessuch as prurigo nodularis, unspecified itch of the elderly as well asother diseases with epithelial barrier dysfunction such as aged skin,inflammatory bowel disease and Crohn's disease, as well as pancreatitis,or of cancer, in particular ovarian cancer, and there is a need forspecific modulators (agonists or inhibitors) thereof.

Human neutrophil elastase (FINE, also know as human leukocyte elastase,FILE) belongs to the chymotrypsin family of serine proteinases. Itscatalytic activity is optimal around pH 7, and the catalytic site iscomposed of three hydrogenbonded amino acid residues: His57, Asp102, andSer195 (in chymotrypsin numbering), which form the so-called catalytictriad. The enzyme is composed of a single peptide chain of 218 aminoacid residues and four disulfide bridges. It shows 30 to 40% sequenceidentity with other elastinolytic or nonelastinolytic serineproteinases. FINE preferentially cleaves the oxidized insulin B chainwith Val at the P1 position, but it also hydrolyzes bonds with Ala, Ser,or Cys in the P1 position.

HNE is located in the azurophilic granules of polymorphonuclearleukocytes (PMNLs), where the HNE concentration is rather high (3 μg ofenzyme/10₆ cells). The major physiological function is to digestbacteria and immune complexes and to take part in the host defenseprocess. HNE aids in the migration of neutrophils from blood to varioustissues such as the airways in response to chemotactic factors. HNE alsotakes part in wound healing, tissue repair, and in the apoptosis ofPMNLs.

In addition to elastin (highly flexible and highly hydrophobic componentof lung connective tissue, arteries, skin, and ligaments), HNE cleavesmany proteins with important biological functions, including differenttypes of collagens, membrane proteins, and cartilage proteoglycans. HNEalso indirectly favours the breakdown of extracellular matrix proteinsby activating procollagenase, prostromelysin, and progelatinase. HNEinactivates a number of endogenous proteinase inhibitors such asα₂-antiplasmin, α₁-antichymotrypsin, antithrombin, and tissue inhibitorof metalloproteinases.

Extracellular elastase activity is tightly controlled in the pulmonarysystem by α1-protease inhibitor (α₁PI), responsible for protection ofthe lower airways from elastolytic damage, whereas the secretoryleukocyte proteinase inhibitor protects mainly the upper airways. In anumber of pulmonary pathophysiological states, e.g., pulmonaryemphysema, chronic bronchitis, and cystic fibrosis, endogenous elastaseinhibitors are inefficient in regulating HNE activity.

HNE is considered to be the primary source of tissue damage associatedwith inflammatory diseases such as pulmonary emphysema, adultrespiratory distress syndrome (ARDS), chronic bronchitis, chronicobstructive pulmonary disease (COPD), pulmonary hypertension, and otherinflammatory diseases as well as bronchopulmonary dysplasia in prematureneonates. HNE is involved in the pathogenesis of increased and abnormalairway secretions commonly associated with airway inflammatory diseases.Thus, bronchoalveolar lavage (BAL) fluid from patients with chronicbronchitis and cystic fibrosis has increased HNE activity. Furthermore,excessive elastase has been proposed to contribute not only to thesechronic inflammatory diseases but also to acute inflammatory diseasessuch as ARDS and septic shock.

Therefore, HNE is considered to be a potential target for the treatmentof diseases involved with HNE activity such as inflammatory diseasessuch as pulmonary emphysema, adult respiratory distress syndrome (ARDS),chronic bronchitis, chronic obstructive pulmonary disease (COPD),pulmonary hypertension, and other inflammatory diseases as well asbronchopulmonary dysplasia in premature neonates, and diseases involvedwith increased and abnormal airway secretions as well as acuteinflammatory diseases. Thus there is a need for specific modulators(agonists or inhibitors) if HNE.

Treatment can be by local or systemic application such a creams,ointments and suppositories or by oral or se or iv application or byinhalation, respectively, in a manner well known in the art.

In one aspect the depsipeptides according to the invention are obtainedby cultivating a Chondromyces crocatus strain which was deposited on 24Apr. 2007 with the DSMZ (DSM 19329) or are obtained by cultivating aChondromyces robustus strain which was deposited on 24 Apr. 2007 withthe DSMZ (DSM 19330) or are obtained by cultivating a Chondromycesapiculatus strain which was deposited on 23 Jun. 2008 with the DSMZ (DSM21595).

The deposit of the strains was made under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor Purposes of Patent Procedure. The deposited strains will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strains are provided merelyas convenience to those skilled in the art and are not an admission thata deposit is required for enablement.

It is to be understood that the present invention is not limited tocultivation of the particular strains Chondromyces crocatus andChondromyces robustus and Chondromyces apiculatus. Rather, the presentinvention contemplates the cultivation of other organisms capable ofproducing depsipeptides, such as mutants or variants of the strains thatcan be derived from this organism by known means such as X-rayirradiation, ultraviolet irradiation, treatment with chemical mutagens,phage exposure, antibiotic selection and the like.

The depsipeptides of the present invention may be biosynthesized byvarious microorganisms. Microorganisms that may synthesize the compoundsof the present invention include but are not limited to bacteria of theorder Myxococcales, also referred to as myxobacteria. Non-limitingexamples of members belonging to the genera of myxobacteria includeChondromyces, Sorangium, Polyangium, Byssophaga, Haploangium, Jahnia,Nannocystis, Koffleria, Myxococcus, Corallococcus, Cystobacter,Archangium, Stigmatella, Hyalangium, Melittangium, Pyxicoccus. Thetaxonomy of myxobacteria is complex and reference is made to Garrity GM, Bell J Y, Lilburn TG (2004) Taxonomic outline of the prokaryotes,Bergey's manual of systematic bacteriology, 2^(nd) edition, release 5.0May 2004. (http://141.150.157.80/bergeysoutline/main.htm).

The compounds of structural formulas (I-XVII) are produced by theaerobic fermentation of a suitable medium under controlled conditionsvia inoculation with a culture of Chondromyces crocatus or Chondromycesrobustus or Chondromyces apiculatus. The suitable medium is preferablyaqueous and contains sources of assimilable carbon, nitrogen, andinorganic salts.

Suitable media include, without limitation, the growth media mentionedbelow in examples 1 and 2. The fermentation is conducted for about 3 toabout 20 days at temperatures ranging from about 10° C. to about 40° C.;however for optimum results it is preferred to conduct the fermentationat about 30° C. The pH of the nutrient medium during the fermentationcan be about 6.0 to about 9.0. The culture media inoculated with thedepsipeptides producing microorganisms may be incubated under aerobicconditions using, for example, a rotary shaker or a stifled tankfermentor Aeration may be achieved by the injection of air, oxygen or anappropriate gaseous mixture to the inoculated culture media duringincubation. As soon as a sufficient amount of the depsipeptide compoundshave accumulated, they may be concentrated and isolated from the culturein conventional and usual manner, for example by extraction- andchromatographic methods, precipitation or crystallization, and/or in amanner disclosed herein. As an example for extraction, the culture canbe mixed and stirred with a suitable organic solvent such as n-butanol,ethyl acetate, cyclohexane, n-hexane, toluene, n-butyl acetate or4-methyl-2-pentanone, the depsipeptide compounds in the organic layercan be recovered by removal of the solvent under reduced pressure. Theresulting residue can optionally be reconstituted with for examplewater, ethanol, methanol or a mixture thereof, and re-extracted with asuitable organic solvent such as hexane, carbon tetrachloride,dichloromethane or a mixture thereof. Following removal of the solvent,the compounds may be further purified for example by chromatographicmethods. As an example for chromatography, stationary phases such assilica gel or aluminia oxide can be applied, with organic elutingsolvents or mixtures thereof, including ethers, ketones, esters,halogenated hydrocarbons or alcohols, or reversed-phase chromatographyon modified silica gel having various functional groups and eluting withorganic solvents or aqueous mixtures thereof, like acetonitrile,methanol or tetrahydrofuran at different pH. Another example ispartition-chromatography, for example in the solid-liquid or in theliquid-liquid mode. Also size exclusion chromatography may be applied,for example using Sephadex LH-20 (Sigma-Aldrich) and eluting withdifferent solvents, preferably with alcohols.

As it is usual in this field, the production as well as the recovery andpurification process may be monitored by a variety of analyticalmethods, including bioassays, TLC, HPLC or a combination thereof, andapplying different detection methods, for TLC typically UV light, iodinevapour or spraying colouring reagents, for HPLC typically UV light, masssensitive or light scattering methods. For example a HPLC technique isrepresented by using a reversed-phase column with a functionalizedsilica gel and applying an eluent which is a linear gradient mixture ofa polar water miscible solvent and water at a specific pH, and adetection method with UV light at different wavelengths and a masssensitive detector.

The depsipetides biosynthesized by microorganisms may optionally besubjected to random and/or directed chemical modifications to formcompounds that are derivatives or structural analogs. Such derivativesor structural analogs having similar functional activities are withinthe scope of the present invention. Depsipeptides may optionally bemodified using methods well-known in the art and described herein.

For instance, derivatives of the depsipeptides of the invention may beprepared by derivatization of cyclic depsipeptides of formula

which comprisesa)—the preparation of compounds wherein A4 is

by treatment of a compound wherein A4 is

with an organic or inorganic acid, e.g. trifluoro acetic acid, sulphuricacid, hydrochloric acid, or a Lewis acid, e.g. borontrifluoride etheratein a solvent, e.g. dichloromethane, THF, or without a solvent at atemperature between −78° C. and 150° C., preferentially between −30° C.and room temperature.b)—the preparation of compounds wherein A4 is

by treatment of a compound wherein A4 is

with molecular hydrogen or source thereof, e.g. cyclohexene, ammoniumformate, in presence of a catalyst e.g. palladium in a solvent e.g.2-propanol at a temperature between −50 and 100° C., preferentially atroom temperature.c)—the preparation of compounds wherein A4 is

by treatment of a compound wherein A4 is

with an organic or inorganic acid, e.g. sulphuric acid, hydrochloricacid or a Lewis acid, e.g. borontrifluoride etherate in presence of anreducing agent, e.g. triethylsilane, a solvent, e.g. dichloromethane,THF, or without a solvent at a temperature between −78° C. and 150° C.,preferentially between −50° C. and room temperature.d)—the preparation of compounds wherein A4 is

by treatment of a compound wherein A4 is

with an substituted or unsubstituted alkanol and an organic or inorganicacid, e.g. trifluoroacetic acid, sulphuric acid, hydrochloric acid, or aLewis acid, e.g. metal salts in a solvent, e.g. substituted andunsubstituted alkanoles, THF, dichloromethane, preferentiallysubstituted and unsubstituted alkanoles, or without a solvent at atemperature between −78° C. and 150° C., preferentially between −30° C.and 50° C.

e)—the preparation of compounds wherein A1 iswherein n=1,2 and A4 is

wherein R preferably is H, alkyl, substituted alkyl, by treatment of acompound wherein A1 is Gln or Asn and A4 is

wherein R preferably is H, alkyl, substituted alkyl, with an substitutedor unsubstituted alkanol and an organic or inorganic acid, e.g.trifluoroacetic acid, sulphuric acid, hydrochloric acid, or a Lewisacid, e.g. borontrifluoride etherate in a solvent, e.g. substituted andunsubstituted alkanols, THF, dichloromethane, preferentially substitutedand unsubstituted alkanols, or without a solvent at a temperaturebetween −78° C. and 150° C., preferentially between −30° C. and roomtemperature.f)—the preparation of compounds wherein A1 is

wherein n=1,2 and A4 is

wherein R preferentially is H, OH, O-alkyl, substituted O-alkyl, O-acyl,by treatment of a compound wherein A1 is Gln or Asn and A4 is

with an dehydrating agent e.g. trifluoroacetic acid anhydride inpresence of a base e.g. diisopropylethylamine (DIPEA), in a solvent e.g.dichloromethane or without a solvent at a temperature between −78° C.and 150° C., preferably between −30° C. and room temperature.g)—the preparation of compounds wherein A4 is

and A6 is

wherein R preferably is alkyl, substituted alkyl, acyl, alkoxycarbonylby treatment of a compound wherein A4 is

and A6 is Tyrwith an alkylating agent e.g. methyl iodide, benzyl bromide, proargylbromide or an acylating agent e.g. ethyl chloroformate or an alkyl oraryl isocyanate in presence of a base e.g. sodium carbonate in a solvente.g. MIT or without a solvent at a temperature between −78° C. and 150°C., preferentially between −30° C. and room temperature, preferablypromoted by ultrasound.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,IC₅₀ and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the present specification and attached claims areapproximations. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of significant figures and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set in the examples, Tables and Figures are reported asprecisely as possible. Any numerical values may inherently containcertain errors resulting from variations in experiments, testingmeasurements, statistical analyses and such. Unless otherwise defined,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

EXAMPLES Example 1 Production of the Compounds Example 1.1 Production ofthe Compounds of Formula (II)-(VII), (XI)-(XIV) and (XVII)

Strain: The Chondromyces crocatus strain was isolated from anenvironmental sample, rotten wood of a walnut tree, in our laboratories.

The strain has been unambiguously identified as a Chondromyces crocatusbased on the morphology of the fruiting bodies as well as on the partialsequence of the 16S-RNA gene. C. crocatus was assigned to biologicalrisk group 1 by the DSMZ (DSMZ (2007)). Chondromyces is a genus in thefamily Polyangiaceae, which belongs to the order Myxococcales within theDelta-proteobacteria. Bacteria of the order Myxococcales, also calledmyxobacteria, are gram-negative rod-shaped bacteria with twocharacteristics distinguishing them from most other bacteria. They swarmon solid surfaces using an active gliding mechanism and aggregate toform fruiting bodies upon starvation (Kaiser (2003)).

The Chondromyces crocatus strain of the invention has been deposited atthe DSMZ under the accession number 19329.

The Chondromyces crocatus strain of the invention is not viable as apure culture and cannot be maintained without a companion strain. Thecompanion strain can be obtained and maintained as a pure culture bystreaking an aliquot of a fermentation co-culture on agar plates (LBmedium). A similar observation was made by the Reichenbach group(Jacobi, et al. (1996), Jacobi, et al. (1997)). Based on a partial DNAsequence of the 16S-rRNA gene of the companion strain of Chondromycescrocatus of this invention, the closest match is Bosea thiooxidans fromthe order Rhizobiales within the Alpha-proteobacteria. The 424 bpsequence fragment 16S-rRNA investigated has about 98% identity (at least8 nucleotide exchanges) to sequence AF508112 (B. thiooxidans) fromgenebank. B. thiooxidans was isolated from soil samples collected fromdifferent agricultural fields around Calcutta, India. It is capable tooxidate reduced inorganic sulfur compounds in the presence of someorganic substrates and was described as a novel species and a novelgenus in 1996 (Das, et al. (1996)). A phylogenetic tree derived from thepartial 16S-RNA sequences of all 5 described Bosea species indicates aseparate position for the Bosea companion strain isolated from C.crocatus.

Cultivation: 100 L fermentor cultures were performed according to thefollowing protocol: Precultures were started by inoculation of 5 ml(=10%) from a liquid culture of Chondromyces crocatus strain of theinvention into 50 ml of medium MD1 (adapted after Bode et al. 2003, seetable 6) in a 200-ml baffled shake flask. After 11 clays incubation at30° C. and 120 rpm on a rotary shaker a 1st intermediate culture wasstarted by inoculation of 10 ml each (=10%) from the preculture into5×100 ml of medium MD1 in 500-ml baffled shake flasks. After 7 claysincubation at 30° C. and 120 rpm on a rotary shaker a 2nd intermediateculture was started by inoculation of 25 ml each (=5%) from the 1stintermediate culture into 19×500 ml of medium MD1 in 2-L nonbaffledshake flasks. After 6 days of incubation at 30° C. and 150 rpm on arotary shaker the whole 2nd intermediate culture (9.5 liters=9.5%) wasused to inoculate 100 liters of production medium POL1. (adapted afterKunze et al. 1995, see table 7)

This 100-L main culture was performed in a 100-L scale steel tankfermentor. Temperature was controlled at 30° C., aeration was 20 l/min(=0.2 vvm) and agitation speed was 50 rpm. A slight overpressure of 0.5bar was maintained inside of the fermentor vessel. Culture pH wasmaintained at 6.9-7.1 by controlled addition of 3N H₂SO₄ or 3N NaOH.After a lag-phase of about 1 day oxygen consumption accelerated forabout 4 days indicating exponential growth of the culture. During thelast 2 days oxygen consumption was slightly reduced indicating astationary phase of the culture. After 7 days the culture was harvestedwith a titer of 5.3 mg/l of a cyclic depsipeptide according to FormulaII.

Extraction: The whole fermentation broth was transferred into a 1600 lsteel vessel and decanted for 1 hour. The wet cell pellet (200 g) washarvested from the bottom fraction by filtration through a paper filter.The cell pellet was extracted 3 times by turaxing it 30 minutes eachwith 10 l ethyl acetate. Then the residual water was separated from thesolvent phase. The solvent phase was washed with 5 l water and thenevaporated to obtain a dry extract referred to as ‘cell extract’. Theculture filtrate was extracted with 200 l ethyl acetate. After 2 hourscontact time, including 1 hour of turaxing, the organic phase wasseparated, washed with 20 l water and finally evaporated to obtain a dryextract referred to as ‘culture filtrate extract’.

Compound isolation: The culture filtrate extract (4.4 g) was dissolvedin 80 mL Methanol. The insoluble ingredients were removed bycentrifugation and the supernatant was evaporated to dryness yielding in3.3 g extract. The extract was dissolved in 7.5 mL MeOH, 3 mL DMSO and0.5 mL dichloromethane and purified by reversed phase chromatography(Waters Sunfire RP18 10 μm, 30×150 mm) using 0.01% formic acid (solventA), and acetonitrile containing 0.1% formic acid (solvent B) as solventsThe flow rate was 50 mL/min. The gradient is shown in Table 1. Thematerial was purified in 7 chromatographic runs. From each run thecollected fractions were analyzed by HPLC, fractions containing thecyclic depsipeptide according to the invention were combined andevaporated in vacuum to dryness. The chromatography yielded in 134 mgcyclic depsipeptide according to formula (II) with a purity of >97% and80 mg with a purity of 90%.

TABLE 1 HPLC gradient used for purification of the cyclic depsipeptideaccording to formula (II) time (min) solvent A (%) solvent B (%) 0.0 9010 1.0 90 10 23.0 50 50 23.1 0 100 27.0 0 100 27.1 90 10 30.0 90 10

TABLE 2 Gradient used for normal phase separation time (min) cyclohexane(%) ethyl acetate (%) methanol (%) 0 75 25 0 10 75 25 0 33 25 75 0 56 2070 10 79 0 50 50 93 0 50 50

The cell extract (6.67 g) was dissolved in dichloromethane/methanol 4:1.The solution was filtered and the filtrate was adsorbed on diatom (2 gdiatom/1 g extract, Isolute®, International Sorbent Technology Ltd.,Hengoed Mid Glam, UK) followed by evaporation. The solid residue wasloaded on a pre-packed silica gel column (4×18 cm, 90 g silica gel40-63) and eluted with a gradient of cyclohexane, ethyl acetate andmethanol. The gradient is shown in Table 2, the flow rate was 28 ml/min.Fractions volumes of 28 ml were collected. The fractions were combinedaccording to the peaks visible in the UV-trace yielding in 12 pooledfractions (A-L). Fractions containing the depsipeptides (H-J) werefurther purified using reversed-phase chromatography. Thechromatographic method and work up procedure is identical to thepurification method described for the culture filtrate. In total 46.1 mgcyclic depsipeptide according to formula (II), 17.9 mg cyclicdepsipeptide according to formula (III) and 6.1 mg of a 1:1 mixture ofthe depsipetides according to formula (VI) and (VII) have been isolated.The assignment of the structures of compound (VI) and (VII) is based onhigh resolution MS and the comparison of the ¹H-NMR data of the mixtureof compound (VI) and (VII) with the ¹H-NMR data of compound (II).

Other cyclic depsipeptide according to formula (II) have also been foundat a lesser concentration in the cell extract. Among these other cyclicdepsipeptides were those according to formula (IV), (V) and (XI)-(XV)and (XVII).

Characterization of Compounds:

Physical Data of Compound of Formula (II)

IR (KBr pellet): 3337, 3297, 3062, 2966, 2936, 2877, 1736, 1659, 1533,1519, 1464, 1445, 1410, 1385, 1368, 1249, 1232, 1205, 989, 832 cm⁻¹

FT-MS (9.4 T APEX-III): 951.5165; calc. for C₄₆H₇₂N₅O₁₂+Na: 951.5162

¹H NMR (600 MHz, d₆-DMSO) δ_(H): −0.10 (3H, d, J=7.0 Hz), 0.65 (4H, m),0.78 (3H, d, J=7.0 Hz), 0.82 (3H, t, J=7.2 Hz), 0.85 (3H, d, J=7.0 Hz),0.89 (3H, d, J=7.0 Hz), 1.02 (1H, m), 1.03 (6H, 2×d, J=7.0 Hz), 1.10(1H, m), 1.21 (3H, d, J=7.0 Hz), 1.25 (1H, m), 1.40 (1H, m), 1.52 (1H,m), 1.76 (6H, m), 1.84 (1H, m), 1.93 (1H, m), 2.15 (2H, m), 2.48 (1H,m), 2.59 (1H, m), 2.69 (1H, m), 2.72 (3H, s), 3.17 (1H, m), 4.32 (2H,m), 4.44 (2H, m), 4.64 (1H, d, J=9.5 Hz), 4.71 (1H, m), 4.94 (1H, s),5.06 (1H, m), 5.49 (1H, m), 6.08 (1H, d, J=2.2 Hz), 6.65 (2H, d, J=8.4),6.74 (1H, s), 7.00 (2H, d, J=8.4 Hz), 7.27 (1H, s), 7.36 (1H, d, J=9.5Hz), 7.66 (1H, d, J=10.2 Hz), 7.74 (1H, d, J=8.8 Hz), 8.02 (1H, d, J=8.1Hz), 8.43 (1H, d, J=8.1 Hz), 9.19 (1H, s).

¹³C NMR (150 MHz) d₆-DMSO δ_(C): 10.35, CH₃; 11.22, CH₃; 13.79, CH₃;16.00, CH₃; 17.63, CH₃; 19.49, 2×CH₃; 20.83, CH₃; 21.72, CH₂; 23.30,CH₃; 23.70, CH₂; 24.16, CH; 24.41, CH₂; 27.35, CH₂; 29.74, CH₂; 30.07,CH₃; 31.44, CH₂; 33.13, CH; 33.19, CH₂; 33.68, CH; 37.39, CH; 39.05,CH₂; 48.75, CH; 50.59, CH; 52.01, CH; 54.11, CH; 54.65, CH; 55.24, CH;60.60, CH; 71.86 CH; 73.89, CH; 115.28, 2×CH; 127.31, Cq; 130.35, 2×CH;156.25, Cq; 169.09, Cq; 169.25, Cq; 169.34, Cq; 169.74, Cq; 170.60, Cq;172.41, Cq; 172.52, Cq; 173.78, Cq; 176.32, Cq

Physical Data of Compound of Formula (III)

FT-MS (9.4 T APEX-III). Found: 965.5318; calc. for C₄₇H₇₄N₈O₁₂+Na:965.5318

¹H NMR (600 MHz) d₆-DMSO δ_(H): −0.10 (3H, d, J=7.0 Hz), 0.64 (4H, m),0.78 (3H, d, J=7.0 Hz), 0.82 (3H, t, J=7.0 Hz), 0.83 (3H, t, J=7.3 Hz),0.85 (3H, d, J=7.0 Hz), 0.89 (3H, d, J=7.0 Hz), 1.01 (3H, d, J=7.1 Hz),1.04 (1H, m), 1.10 (1H, m), 1.21 (3H, d, J=7.0 Hz), 1.25 (1H, m), 1.32(1H, m), 1.40 (1H, m), 1.53 (2H, m), 1.77 (6H, m), 1.84 (1H, m), 1.92(1H, m), 2.12 (1H, m), 2.16 (1H, m), 2.28 (1H, m), 2.59 (1H, m), 2.68(1H, m), 2.72 (3H, s), 3.17 (1H, m), 4.32 (1H, m), 4.38 (1H, m), 4.43(1H, d, J=10.2 Hz), 4.46 (1H, m), 4.63 (1H, d, J=9.5 Hz), 4.71 (1H, m),4.94 (1H, m), 5.06 (1H, m), 5.49 (1H, m), 6.11 (1H, s, broad), 6.65 (2H,d, J=8.8 Hz), 6.73 (1H, s), 7.00 (2H, d, J=8.8 Hz), 7.27 (1H, s), 7.37(1H, d, J=9.5 Hz), 7.66 (1H, d, J=10.2 Hz), 7.75 (1H, d, J=9.7 Hz), 8.07(1H, d, J=8.1 Hz), 8.45 (1H, d, J=8.8 Hz), 9.24 (1H, broad)

Physical Data of Compound of Formula (IV)

FT-MS (9.4 T APEX-III). Found: 947.5196; calc. for C₄₇H₇₂N₈O₁₁+Na:947.5213

¹H NMR (600 MHz) d₆-DMSO δ_(H): 0.08 (3H, d, J=7.0 Hz), 0.68 (3H, t,J=7.2 Hz), 0.71 (3H, d, J=7.0 Hz), 0.78 (3H, d, J=7.0 Hz), 0.83 (3H, t,J=7.3 Hz), 0.84 (1H, m), 0.87 (3H, t, J=7.2 Hz), 0.88 (3H, d, J=7.0 Hz),0.99 (3H, d, J=7.1 Hz), 1.08 (1H, m), 1.17 (3H, d, J=6.7 Hz), 1.18 (1H,m), 1.31 (2H, m), 1.43 (1H, m), 1.51 (1H, m), 1.54 (1H, m), 1.76 (2H,m), 1.90 (1H, m), 1.94 (1H, m), 2.01 (1H, m), 2.10 (1H, m), 2.16 (1H,m), 2.26 (1H, m), 2.46 (2H, m), 2.73 (1H, m), 2.74 (3H, s), 3.19 (1H,m), 4.34 (1H, m), 4.36 (1H, m), 4.51 (1H, m), 4.55 (1H, m), 4.66 (1H, d,J=10.0 Hz), 4.79 (1H, d, J=11.0 Hz), 5.19 (1H, m), 5.28 (1H, m), 5.44(1H, m), 6.25 (1H, d, J=7.3 Hz), 6.33 (1H, d, J=8.8 Hz), 6.68 (2H, d,J=8.8 Hz), 6.75 (1H, s), 7.04 (2H, d, J=8.8 Hz), 7.28 (1H, s), 7.32 (1H,d, J=8.8 Hz), 7.91 (1H, d, J=9.5 Hz), 8.05 (1H, d, J=8.1 Hz), 8.57 (1H,d, J=8.9 Hz), 9.38 (1H, broad)

Physical Data of Compound of Formula (V)

FT-MS (9.4 T APEX-III). Found: 933.5053; calc. for C₄₆H₇₀N₈O₁₁+Na:953.5056

¹H NA/IR (600 MHz) d₆-DMSO δ_(H): 0.08 (3H, d, J=7.0 Hz), 0.68 (3H, t,J=7.2 Hz), 0.71 (3H, d, J=7.0 Hz), 0.79 (3H, d, J=7.0 Hz), 0.83 (1H, m),0.88 (3H, t, J=7.2 Hz), 0.89 (3H, d, J=7.0 Hz), 1.01 (3H, d, J=7.0 Hz),1.03 (3H, d, J=7.0 Hz) 1.08 (1H, m), 1.17 (3H, d, J=6.7 Hz), 1.20 (1H,m), 1.31 (1H, m), 1.42 (1H, m), 1.54 (1H, m), 1.74 (2H, m), 1.91 (2H,m), 2.02 (1H, m), 2.10 (1H, m), 2.15 (1H, m), 2.46 (3H, m), 2.75 (3H,s), 2.76 (1H, m), 3.19 (1H, m), 4.32 (1H, m), 4.34 (1H, m), 4.51 (1H,m), 4.55 (1H, m), 4.66 (1H, d, J=9.5 Hz), 4.79 (1H, d, J=11.0 Hz), 5.19(1H, m), 5.28 (1H, m), 5.43 (1H, m), 6.25 (1H, d, J=7.0 Hz), 6.33 (1H,d, J=8.5 Hz), 6.68 (2H, d, J=8.8 Hz), 6.75 (1H, s), 7.04 (2H, d, J=8.8Hz), 7.28 (1H, s), 7.31 (1H, d, J=8.8 Hz), 7.90 (1H, d, J=9.5 Hz), 7.99(1H, d, J=8.1 Hz), 8.52 (1H, d, J=8.8 Hz), 9.30 (1H, broad)

Physical Data of Compound of Formula (XI)

ESI-MS: pos. mode: m/z=951.5 (M+Na), neg. Mode: m/z=927.5 (M−H);monoisotopic MW 928.5, C₄₆H₇₂N₈O₁₂

¹H NMR (600 MHz) d₆-DMSO δ_(H): −0.11 (3H, d, J=6.6 Hz), 0.64 (4H, m),0.77 (3H, d, J=6.6 Hz), 0.83 (3H, m), 0.85 (3H, m), 0.87 (3H, m), 0.89(3H, m), 1.01 (3H, m), 1.10 (1H, m), 1.21 (3H, d, J=5.9 Hz), 1.32 (1H,m), 1.40 (1H, m), 1.52 (2H, m), 1.75 (5H, m), 1.84 (1H, m), 1.91 (1H,m), 2.05 (1H, m), 2.15 (2H, m), 2.27 (1H, m), 2.59 (1H, m), 2.68 (1H,m), 2.73 (3H, s), 3.16 (1H, m), 4.31 (1H, m), 4.37 (1H, m), 4.43 (1H,m), 4.45 (1H, m), 4.63 (1H, d, J=8.8 Hz), 4.69 (1H, m), 4.94 (1H, m),5.05 (1H, m), 5.50 (1H, m), 6.15 (1H, s, broad), 6.65 (2H, d, J=8.1 Hz),6.75 (1H, s), 7.00 (211, d, J=8.1 Hz), 7.29 (1H, s), 7.37 (1H, d, J=9.5Hz), 7.64 (1H, d, J=9.5 Hz), 7.78 (1H, d, J=8.8 Hz), 8.09 (1H, d, J=8.1Hz), 8.49 (1H, d, J=9.5 Hz), (OH of tyrosine not visible)

Physical Data of Compound of Formula (XII)

ESI-MS: pos. mode: m/z=923.5 (M+Na), neg. Mode: m/z=899.5 (M−H);monoisotopic MW 900.5, C₄₄H₆₈N₈O₁₂.

¹H NMR (500 MHz) d₆-DMSO δ_(H): −0.11 (3H, d, J=6.4 Hz), 0.63 (4H, m),0.75 (3H, d, J=6.4 Hz), 0.83 (6H, d, J=7.0 Hz), 0.87 (3H, d, J=6.4 Hz),1.03 (1H, m), 1.10 (1H, m), 1.20 (3H, d, J=6.4 Hz), 1.25 (1H, m), 1.38(1H, m), 1.50 (1H, m), 1.73 (1H, m), 1.75 (2H, m), 1.77 (1H, m), 1.79(3H, m), 1.85 (3H, s), 1.85 (1H, m), 2.12 (1H, m), 2.16 (1H, m), 2.55(1H, m), 2.67 (1H, m), 2.70 (3H, s), 3.13 (1H, m), 4.30 (1H, m), 4.40(1H, m), 4.43 (2H, m), 4.59 (1H, d, J=9.5 hz), 4.71 (1H, m), 4.92 (1H,m), 5.02 (1H, m), 5.46 (1H, m), 6.08 (1H, s, broad), 6.62 (2H, d, J=8.5Hz), 6.71 (1H, s), 6.97 (2H, d, J=8.5 Hz), 7.22 (1H, s), 7.34 (1H, d,J=9.2 Hz), 7.64 (1H, d, J=9.5 Hz), 7.93 (1H, d, J=9.2 Hz), 8.06 (1H, d,J=7.6 Hz), 8.39 (1H, d, J=8.5 Hz), 9.06 (1H, s, broad)

Physical Data of Compound of Formula (XIII)

FT-MS (9.4 T APEX-III). Found: 961.5039; calc. for C₄₉H₇₀N₈O₁₂+Na:985.5005

¹H NMR (500 MHz) d₆-DMSO δ_(H): −0.12 (3H, d, J=6.4 Hz), 0.62 (4H, m),0.75 (3H, d, J=6.4 Hz), 0.81 (4H, m), 0.87 (3H, d, J=6.4 Hz), 1.08 (1H,m), 1.20 (3H, m), 1.22 (3H, m), 1.38 (1H, m), 1.46 (1H, m), 1.50 (1H,m), 1.71 (1H, m), 1.73 (2H, m), 1.76 (2H, m), 1.82 (1H, m), 1.94 (1H,m), 2.01 (1H, m), 2.23 (2H, m), 2.56 (1H, m), 2.66 (1H, m), 2.69 (3H,s), 3.13 (1H, m), 4.30 (1H, m), 4.41 (2H, m), 4.55 (1H, m), 4.65 (1H, d,J=9.5 Hz), 4.70 (1H, m), 4.92 (1H, m), 5.04 (1H, m), 5.48 (1H, m), 6.09(1H, s, broad), 6.64 (2H, d, J=8.5 Hz), 6.81 (1H, s), 6.97 (2H, d, J=8.5Hz), 7.33 (1H, s), 7.35 (1H, d, J=9.2 Hz), 7.46 (2H, t, J=7.3 Hz), 7.53(1H, t, J=7.3 Hz), 7.66 (1H, d, J=9.5 Hz), 7.88 (2H, d, J=7.3 Hz), 7.95(1H, d, J=9.5 Hz), 8.46 (1H, d, J=8.5 Hz), 8.71 (1H, d, J=7.3 Hz); (OHof tyrosine not visible)

Physical Data of Compound of Formula (XIV)

ESI-MS: pos. mode: m/z=927.5 (M+H), neg. Mode: m/z=925.5 (M−H);monoisotopic MW 926.5, C₄₇H₇₄N₈O₁₁

¹H NMR (500 MHz) d₆-DMSO δ_(H): 0.00 (1H, m), 0.48 (3H, t, J=7.5 Hz),0.70 (3H, d, J=7.0 Hz), 0.73 (3H, t, J=7.0 Hz), 0.76 (3H, d, J=7.3 Hz),0.79 (3H, t, J=7.3 Hz), 0.83 (611, d, J=6.4 Hz), 0.89 (1H, m), 0.95 (1H,m), 0.98 (3H, d, J=7.0 Hz), 1.04 (3H, d, J=6.1 Hz), 1.09 (1H, m), 1.17(1H, m), 1.28 (1H, m), 1.31 (1H, m), 1.36 (1H, m), 1.38 (1H, m), 1.55(1H, m), 1.61 (1H, m), 1.68 (1H, m), 1.79 (1H, m), 1.82 (1H, m), 1.95(2H, m), 2.15 (3H, m), 2.25 (1H, m), 2.66 (3H, s), 2.76 (1H, m), 3.14(1H, m), 3.40 (1H, m), 3.42 (1H, m), 4.33 (1H, m), 4.36 (1H, m), 4.45(2H, m), 4.55 (2H, m), 4.69 (1H, m), 5.06 (1H, m), 6.63 (2H, d, J=8.2Hz), 6.71 (1H, s, broad), 7.01 (2H, d, J=8.2 Hz), 7.27 (1H, s, broad),7.36 (1H, d, J=9.5 Hz), 8.00 (1H, d, J=9.5 Hz), 8.17 (1H, d, J=4.00 Hz),8.22 (1H, d, J=7.3 Hz), 8.53 (1H, d, J=9.5 Hz), 9.14 (1H, s, broad)

Physical Data of Compound of Formula (XVII)

ESI-MS: pos. mode: m/z=985.4 (M+Na), neg. Mode: m/z=961.5 (M−H);monoisotopic MW 962.5, C₄₅H₇₀N₈O₁₃S

¹H NMR (600 MHz) d₆-DMSO δ_(H):). δ_(H): no assignment of chemicalshifts (mixture of two diastereomers, structure assignment troughcomparison with other related compounds, e.g. compound (II)

Example 1.2 Production of Compound of Formula (VIII, IX, X)

Strain: The Chondromyces robustus strain was isolated from a dungsample. The Chondromyces robustus strain of the invention has beenidentified as a Chondromyces robustus based on the morphology of thefruiting bodies as well as on the partial sequence of the 16S-RNA gene.C. robustus was assigned to biological risk group 1 by the DSMZ (DSMZ(2007)). Chondromyces is a genus in the family Polyangiaceae, whichbelongs to the order Myxococcales within the Delta-proteobacteria.Bacteria of the order Myxococcales, also called myxobacteria, aregram-negative rod-shaped bacteria with two characteristicsdistinguishing them from most other bacteria. They swarm on solidsurfaces using an active gliding mechanism and aggregate to formfruiting bodies upon starvation (Kaiser (2003)).

The Chondromyces robustus strain of the invention has been deposited atthe DSMZ under the accession number 19330.

Cultivation: 100 L fermentor cultures were performed according to thefollowing protocol:

Precultures were started by inoculation of 20 ml each (=20%) from aliquid culture of the Chondromyces robustus strain of the invention into6×100 ml of medium MD1 (adapted after Bode et al. 2003) in 500-mlbaffled shake flasks. After 1 day of incubation at 30° C. and 120 rpm ona rotary shaker a 1^(st) intermediate culture was started by inoculationof 100 ml each (=25%) from the preculture into 6×400 ml of medium MD1 in2-L baffled shake flasks. After 3 days incubation at 30° C. and 120 rpmon a rotary shaker a 2^(nd) intermediate culture was started byinoculation of 3 liters (=20%) from the 1^(st) intermediate culture intoa 20-L steel tank fermentor containing 15 liters of medium MD1.Temperature was controlled at 30° C., aeration was 20 l/min (=1.0 vvm)and agitation speed was 80 rpm. A slight overpressure of 0.5 bar wasmaintained inside of the fermentor vessel. Although there was no pHcontrol the pH of the culture decreased only slightly from pH 6.95 atstart to pH 6.88 on day 7. After 7 days the whole 2^(nd) d intermediateculture (18 liters=20%) was used to inoculate 90 liters of productionmedium POL1 (adapted after Kunze et al. 1995) (starting volume=108liters). The main culture was performed in a 100-L scale steel tankfermentor. Temperature was controlled at 30° C., aeration was 30 l/min(=0.3 vvm) and agitation speed was in the beginning 50 rpm and after 4days 80 rpm. A slight overpressure of 0.5 bar was maintained inside ofthe fermentor vessel. Culture pH was maintained at 6.8-7.2 by controlledaddition of 2N HSO₄ or 1.5N NaOH. After 14 days the culture washarvested with a titer of 3 mg/1

Extraction: The whole fermentation broth was transferred into a 1600 lsteel vessel and decanted for 1 hour. The wet cell pellet (about 200 g)was harvested from the bottom fraction by filtration through a paperfilter. The cell pellet was extracted 3 times by turaxing it 30 minuteseach with 10 l ethyl acetate. Then the residual water was separated fromthe solvent phase. The solvent phase was washed with 5 l water and thenevaporated to obtain 11.9 g dry extract referred to as ‘cell extract’.

The culture filtrate was extracted with 200 l ethyl acetate. After 2hours contact time, including 1 hour of turaxing, the organic phase wasseparated, washed with 20 l water and finally evaporated to obtain 12.5g of dry extract referred to as ‘culture filtrate extract’.

Compound isolation: Each extract (from mycelium and culture filtrate)was dissolved in dichloromethane/methanol 4:1. The solution was filteredand the filtrate was adsorbed on diatom (2 g diatom/1 g extract,Isolute®, International Sorbent Technology Ltd., Hengoed Mid Glam, UK)followed by evaporation. The solid residue was loaded on a pre-packedsilica gel column (4×18 cm, 100 g silica gel 40-63) and eluted with agradient of cyclohexane, ethyl acetate and methanol. The gradient isshown in Table 4, the flow rate was 28 ml/min. Fractions volumes of 28ml were collected. The fractions were combined according to the peaksvisible in the UV-trace. The fraction containing the cyclic depsipeptideof the invention was further purified using reversed-phasechromatography (Waters Sunfire RP18 10 μm, 30×150 mm) using 0.01% formicacid (solvent A), and acetonitrile containing 0.1% formic acid (solventB) as solvents. The flow rate was 50 mL/min. The gradient is shown inTable 5. For injection the material was dissolved in MeOH/DMSO 1:1(concentration 200 mg/mL). The collected fractions were analyzed byHPLC, fractions containing the cyclic depsipeptide of the invention werecombined and evaporated in vacuum to dryness. The chromatography of theextract yielded in 52 mg pure (>97%) cyclic depsipeptide according toformula (VIII) A total of 85 mg pure cyclic depsipeptide according toformula (VIII) could be isolated from the combined extracts.

Other cyclic depsipeptide according to formula (VIII) have also beenfound at a lesser concentration in the cell extract. Among these othercyclic depsipeptides were those according to formula (IX) and (X).

TABLE 4 Gradient used for normal phase separation time (min) cyclohexane(%) ethyl acetate (%) methanol (%) 10 75 25 0 33 25 75 0 56 20 70 10 790 50 50 93 0 50 50

TABLE 5 HPLC gradient used for purification of cyclic depsipeptideaccording to formula (VIII) time (min) solvent A (%) solvent B (%) 0.075 25 1.0 75 25 23.0 55 45 23.1 0 100 27.0 0 100 27.1 75 25 30.0 75 25Media (Adjusted to pH 7.0 with 50 mM HEPES)

TABLE 6 MD1 (pre-culture medium) Concentration Substance [g/L] Casitone3 CaCl₂ × 2 H₂O 0.5 MgSO₄ × 7 H₂O 2 D(+)-Glucose water free 1Cyanocobalamine 0.5 mg Antifoam B 0.2 mL Ferrioxamine solution [100ng/mL] 1 mL

TABLE 7 POL1 (production medium) Concentration Substance [g/L] Potatoprotein 4 Soluble starch 3 CaCl₂ × 2 H₂O 0.5 MgSO₄ × 7 H₂O 2Cyanocobalamine 0.25 mg HEPES 12 Standard Trace Element Solution 1901 1mL XAD16 35Characterization of Compounds:Physical Data of Compound of Formula (VIII)

FT-MS (9.4 T APEX-III). Found: 985.5007; calc. for C₄₉H₇₀N₈O₁₂+Na:985.5005.

¹H NMR (600 MHz) d₆-DMSO δ_(H): 0.74 (6H, d, J=7.0 Hz), 0.85 (3H, d,J=7.0 Hz), 0.88 (3H, d, J=7.0 Hz), 0.89 (6H, d, J=7.0 Hz), 1.18 (3H, d,J=6.7 Hz), 1.32 (1H, m), 1.46 (1H, m), 1.57 (2H, m), 1.72 (3H, m), 1.81(1H, m), 1.88 (1H, m), 1.98 (1H, m), 2.02 (2H, m), 2.11 (3H, m), 2.42(1H, m), 2.73 (1H, m), 2.77 (3H, s), 2.87 (1H, m), 3.12 (1H, m), 3.64(1H, m), 4.23 (1H, m), 4.40 (1H, m), 4.58 (1H, d, J=9.5 Hz), 4.75 (2H,m), 4.93 (1H, m), 5.07 (1H, s), 5.40 (1H, m), 6.03 (1H, s), 6.74 (1H,s), 6.79 (2H, d, J=8.4 Hz), 6.84 (2H, d, J=7.8 Hz), 7.02 (2H, d, J=8.4Hz), 7.10 (1H, d, J=9.3 Hz), 7.14 (1H, t, J=7.8 Hz), 7.19 (2H, t, J=7.8Hz), 7.26 (1H, s), 7.42 (1H, d, J=9.8 Hz), 7.89 (1H, d, J=9.2 Hz), 8.03(1H, d, J=7.9 Hz), 8.38 (1H, d, J=8.9 Hz), 9.40 (1H, s)

¹³C NMR (150 MHz) d₆-DMSO δ_(C): 17.13, CH₃; 17.63, CH₃; 19.32, CH₃;20.90, CH₃; 21.64, CH₂; 22.34, CH₃; 22.34, CH₃; 23.32, CH₃; 24.10, CH;25.63, CH; 27.63, CH₂; 29.30, CH₂; 30.37, CH₃; 30.86, CH; 31.52, CH₂;32.83, CH₂; 35.33, CH₂; 38.98, CH₂; 44.42, CH₂; 48.52, CH; 50.19, CH;50.24, CH; 51.99, CH; 54.62, CH; 55.63, CH; 60.90, CH; 71.86 CH; 73.70,CH; 115.32, 2×CH; 126.21, CH; 127.50, Cq; 127.74, 2×CH; 129.42, 2×CH;130.43, 2×CH; 136.72, Cq; 156.23, Cq; 168.93, Cq; 169.18, Cq; 169.18,Cq; 170.18, Cq; 170.39, Cq; 171.72, Cq; 171.96, Cq; 172.50, Cq; 173.82,Cq

Physical Data of Compound of Formula (IX)

FT-MS (9.4 T APEX-III). Found: 969.5058; calc. for C₄₉H₇₀N₈O₁₁+Na;969.5056.

¹H NMR (600 MHz) d₆-DMSO δ_(H):): 0.53 (3H, d, J=6.6 Hz), 0.73 (3H, d,J=6.6 Hz), 0.74 (3H, d, J=6.6 Hz), 0.81 (3H, d, J=6.6 Hz), 0.86 (6H, d,J=6.6 Hz), 1.08 (3H, d, J=6.5 Hz), 1.20 (1H, m), 1.33 (3H, m), 1.52 (1H,m), 1.64 (1H, m), 1.80 (2H, m), 2.01 (1H, m), 2.04 (2H, m), 2.15 (4H,m), 2.25 (1H, m), 2.30 (1H, m), 2.74 (3H, s), 2.83 (1H, m), 3.12 (1H,m), 3.32 (1H, m), 3.38 (1H, m), 4.14 (1H, m), 4.27 (1H, m), 4.40 (1H,m), 4.59 (1H, m), 4.61 (1H, m), 4.94 (1H, m), 4.99 (1H, m), 5.10 (1H,m), 6.42 (2H, d, J=8.8 Hz), 6.75 (1H, s), 7.04 (2H, d, J=8.8 Hz), 7.10(1H, t, J=7.3 Hz), 7.15 (2H, t, J=7.3 Hz), 7.23 (2H, d, J=7.3 Hz), 7.30(1H, s), 7.41 (1H, d, J=9.5 Hz), 8.05 (1H, d, J=9.5 Hz), 8.23 (1H, d,J=8.1 Hz), 8.47 (1H, d, J=4.4 Hz), 8.71 (1H, d, J=10.2 Hz). (signal ofproton of hydroxy group of tyrosine not visible)

Physical Data of Compound of Formula (X)

FT-MS (9.4 T APEX-III). Found: 955.4896; calc. for C₄₈H₆₈N₈O₁₁+Na:955.4900.

¹H NMR (600 MHz) d₆-DMSO □_(H):). □_(H): no assignment of chemicalshifts (mixture of rotameres, assignment of structure based oncomparison of NMR data (missing N-methylgroup) with NMR data of compound(IX).

Example 1.3 Production of Compounds of Formula (XV-XVI)

Strain: The Chondromyces apiculatus strain was isolated from a soilsample. The Chondromyces strain of the invention has been identified asa Chondromyces apiculatus based on the partial sequence of the 16S-RNAgene. C. apiculatus was assigned to biological risk group 1 by the DSMZ(DSMZ (2007)). Chondromyces is a genus in the family Polyangiaceae,which belongs to the order Myxococcales within the Delta-proteobacteria.Bacteria of the order Myxococcales, also called myxobacteria, aregram-negative rod-shaped bacteria with two characteristicsdistinguishing them from most other bacteria. They swarm on solidsurfaces using an active gliding mechanism and aggregate to formfruiting bodies upon starvation (Kaiser (2003)).

The Chondromyces robustus strain of the invention has been deposited atthe DSMZ under the accession number DSM 21595.

Cultivation:

Precultures were started by inoculation of 20 ml each (=20%) from aliquid culture of the Chondromyces apiculatus strain of the inventioninto 10×100 ml of medium MD1 (adapted after Bode et al. 2003) in 500-mlbaffled shake flasks. After 6 days of incubation at 30° C. and 120 rpmon a rotary shaker the cultures with total volume of 1 L weretransferred into a 50 L Wave® bag together with 5 L of medium MD1. After7 days of incubation in a BioWave 200 SPS reactor (Wave Biotec AG,Switzerland) 40 L of medium M7/14 were added to the bag to start theproduction. The culture was harvested after 19 days.

Extraction:

For harvesting the air in the headspace of the wave bag was removed withvacuum and the bag was hang up to allow sedimentation of the resin andthe cells. After 1 hour of sedimentation 43 l of supernatant wereremoved and discarded. The residual 71 containing the cells and theresin were frozen overnight. After thawing, cells and resin were gainedby filtration through a paper filter. The filtrate was discarded. Thecell/resin pellet (wet weight approximately 3 kg) was transferred into ametal vessel and extracted two times with 15 l ethyl acetate, with 5minutes turaxing during the first extraction. The mixtures of bothbatches were separated through a paper filtration and the filtrates thenunified. After separating the organic solvent phase from the water phasethe solvent phase was washed with 2 l of pure water and then evaporateduntil dry. The water phases were discarded.

Compound isolation: The extract (5 g) was dissolved indichloromethane/methanol 4:1. The solution was filtered and the filtratewas adsorbed on diatom (2 g diatom/1 g extract, Isolute®, InternationalSorbent Technology Ltd., Hengoed Mid Glam, UK) followed by evaporation.The solid residue was loaded on a pre-packed silica gel column (4×18 cm,100 g silica gel 40-63) and eluted with a gradient of cyclohexane, ethylacetate and methanol. The gradient is shown in Table 2, the flow ratewas 28 ml/min. Fractions volumes of 28 ml were collected. The fractionswere combined according to the peaks visible in the UV-trace. Thefraction containing the cyclic depsipeptides of the invention werefurther purified using reversed-phase chromatography (Waters SunfireRP18 10 μm, 30×150 mm) using 0.01% formic acid (solvent A), andacetonitrile containing 0.1% formic acid (solvent B) as solvents. Theflow rate was 50 mL/min. The gradient is shown in Table 1. For injectionthe material was dissolved in 1.6 mL MeOH/DMSO 1:1. The collectedfractions were analyzed by HPLC, fractions containing the cyclicdepsipeptides of the invention were combined and evaporated in vacuum todryness. The chromatography of the extract yielded in 7 mg pure cyclicdepsipeptide according to formula (XV) and 1.2 g pure cyclicdepsipeptide according to formula (XVI)

Media

TABLE 8 MD1 (pre-culture medium) Concentration Substance [g/L] Casitone3 CaCl₂ × 2 H₂O 0.5 MgSO₄ × 7 H₂O 2 D(+)-Glucose water free 1Cyanocobalamine 0.5 mg Antifoam B 0.2 mL Ferrioxamine solution [100ng/mL] 1 mL (Adjusted to pH 7.0 with 50 mM HEPES)

TABLE 9 M7/14 (production medium) Concentration Substance [g/L] Yeastextract 1 CaCl₂ × 2 H₂O 1 MgSO₄ × 7 H₂O 1 Potato starch 5 HEPES 12Potato protein 5 D(+)-Glucose water free 2 Cyanocobalamine 0.1 mgAntifoam B 0.2 mL Ferrioxamine solution [100 ng/mL] 3 mL XAD-16 resin 35(Adjusted to pH 7.4)Characterization of Compounds:Physical Data of Compound of Formula (XV)

FT-MS (9.4 T APEX-III). Found: 1014.5272; calc. for C₅₀H₇₃N₉O₁₂+Na:1014.5276.

¹H NMR (600 MHz) d₆-DMSO δ_(H): 0.72 (3H, d, J=6.6 Hz), 0.82 (3H, d,J=6.6 Hz), 0.85 (6H, d, J=6.6 Hz), 1.01 (1H, m), 1.02 (6H, d, J=6.6 Hz),1.17 (3H, d, J=6.6 Hz), 1.21 (1H, m), 1.31 (1H, m), 1.36 (2H, m), 1.42(1H, m), 1.49 (1H, m), 1.54 (1H, m), 1.56 (1H, m), 1.69 (3H, m), 1.79(1H, m), 1.81 (1H, m), 2.40 (1H, m), 2.48 (1H, m), 2.73 (1H, m), 2.76(3H, s), 2.87 (1H, m), 2.91 (1H, m), 2.98 (1H, m), 3.10 (1H, m), 3.63(1H, m), 4.22 (1H, m), 4.42 (1H, td, J=8.1, 5.1 Hz), 4.58 (1H, m), 4.75(2H, m), 4.90 (1H, m), 5.06 (1H, m), 5.38 (1H, m), 5.42 (2H, broad),5.96 (1H, t, J=5.5 Hz), 6.06 (1H, s, broad), 6.78 (2H, d, J=8.1 Hz),6.83 (21-1, d, J=7.3 Hz), 7.00 (2H, d, J=8.1 Hz), 7.10 (1H, d, J=9.5Hz), 7.14 (1H, t, J=7.3 Hz), 7.19 (2H, t, J=7.3 Hz), 7.46 (1H, d, J=9.5Hz), 7.77 (1H, d, J=9.5 Hz), 7.97 (1H, d, J=8.1 Hz), 8.37 (1H, d, J=8.8Hz), 9.50 (1H, s, broad)

Physical Data of Compound of Formula (XVI)

ESI-MS: pos. mode: m/z=1004.4 (M+Na), neg. Mode: m/z=976.5 (M−H);monoisotopic MW 977.5, C₄₉H₇₁N₉O₁₂

¹H NMR (600 MHz) d₆-DMSO δ_(H): 0.72 (6H, d, J=6.6 Hz), 0.85 (3H, d,J=6.6 Hz), 0.87 (3H, d, J=6.6 Hz), 1.01 (6H, d, J=6.6 Hz), 1.17 (3H, d,J=6.6 Hz), 1.30 (1H, m), 1.35 (2H, m), 1.43 (1H, m), 1.48 (1H, m), 1.56(1H, m), 1.57 (1H, m), 1.69 (1H, m), 1.71 (2H, m), 1.79 (1H, m), 2.08(1H, m), 2.41 (1H, m), 2.48 (1H, m), 2.77 (3H, s), 2.87 (1H, m), 2.92(1H, m), 2.98 (1H, m), 3.09 (1H, m), 3.63 (1H, m), 4.22 (1H, m), 4.42(1h, td, J=8.1, 5.1 Hz), 4.57 (1H, m), 4.74 (1, m), 4.76 (1H, m), 491(1H, m), 5.06 (1H, s), 5.39 (1H, m), 5.43 (2H, s, broad), 5.96 (1H, t,J=5.5 Hz), 6.07 (1H, s, broad), 6.78 (2H, d, J=8.1 Hz), 6.84 (2H, d,J=7.3 Hz), 7.00 (21-1, d, J=8.1 Hz), 7.11 (1H, d, J=9.5 Hz), 7.15 (1H,t, J=7.3 Hz), 7.19 (2H, t, J=7.3 Hz), 7.42 (1H, d, J=9.5 Hz), 7.78 (1H,d, J=9.5 Hz), 7.98 (1H, d, J=8.1 Hz), 8.39 (1H, d, J=8.8 Hz), 9.52 (1H,s, broad)

Example 2 Determination of Biological Activity In Vitro

The compounds of the present invention, e.g. including a compound offormula II-X, exhibit pharmacological activity and are therefore usefulas pharmaceuticals. E.g., the compounds of the present invention arefound to inhibit Kallikrein-7 activity and HNE activity.

Compounds of the present invention have IC₅₀ values between 1 nM and 10μM as determined in the following assay:

Example 2.1 Kallikrein-7 Inhibitory Activity In Vitro

Materials and Buffers

The fluorescence-quenched substrateAc-Glu-Asp(EDANS)-Lys-Pro-Ale-Leu-Phe^Arg-Leu-Gly-Lys(DABCYL)-Glu-NH₂(where ^ indicates the scissile bond, identified by MS analysis) ispurchased from Biosyntan (Berlin, Germany) and kept as a 5 mM stocksolution in DMSO at −20° C. All other chemicals are of analytical grade.

Enzymatic reactions are conducted in 50 mM sodium citrate buffer at pH5.6 containing 150 mM NaCl and 0.05% (w/v) CHAPS.

All protein and peptide containing solutions are handled in siliconizedtubes (Life Systems Design, Merenschwand, Switzerland). The compoundsolutions as well as the enzyme and the substrate solutions aretransferred to the 384-well plates (black Cliniplate; cat. no. 95040020Labsystems Oy, Finland) by means of a CyBi-Well 96-channel pipettor(CyBio AG, Jena, Germany).

Instrumentation for FI Measurements

For fluorescence intensity (FI) measurements an Ultra Evolution reader(TECAN, Maennedorf, Switzerland) is used. The instrument is equippedwith a combination of a 350 nm (20 nm bandwidth) and a 500 nm (25 nmbandwidth) bandpass filter for fluorescence excitation and emissionacquisition, respectively. To increase the signal:background ratio, anappropriate dichroic mirror is employed. The optical filters and thedichroic mirror are purchased from TECAN. The fluorophores in each wellare excited by three flashes per measurement.

Determination of IC₅₀ Values

For the determination of IC₅₀ values the assay is performed at roomtemperature in 384-well plates. All final assay volumes were 30 μl. Testcompounds are dissolved in 90% (v/v) DMSO/water and diluted in water(containing 0.05% (w/v) CHAPS) to 3-times the desired assayconcentration. The 11 final compound concentrations are: 0.3 nM, 1 nM, 3nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM and 30 μM. For eachassay, 10 μl water/CHAPS (±test compound) are added per well, followedby 10 μl protease solution (diluted with 1.5× assay buffer). Theprotease concentration in final assay solution is 0.2 nM (according tothe enzyme concentrations determined by the Bradford method). After 1hour of incubation at room temperature, the reaction is started byaddition of 10 μl substrate solution (substrate dissolved in 1.5× assaybuffer, final concentration was 2 μM). The effect of the compound on theenzymatic activity is obtained from the linear progress curves anddetermined from two readings, the first one taken directly after theaddition of substrate and the second one after 1 hour. The IC₅₀ value iscalculated from the plot of percentage of inhibition vs. inhibitorconcentration using non-linear regression analysis software (XLfit,Vers. 4.0; ID Business Solution Ltd., Guildford, Surrey, UK).

The cyclic depsipeptides inhibited hKallikrein7 with IC50 values asindicated in table 11.

TABLE 10 Cyclic depsipeptide Cyclic depsipeptide according to formula(II) according to formula (III) Enzyme IC50 μM IC50 μM hKallikrein70.001 0.0004

TABLE 11 Cyclic depsipeptide human neutrophile hKallikrein7 according toformula: elastase IC 50 [μM] IC 50 [μM] formula II 0.01 0.001 formulaIII 0.01 0.0004 formula IV 0.07 0.005 formula V 0.06 0.006 formula IX0.01 0.001 formula X 0.2 0.02 formula XII 0.05 0.004 formula XIII 0.030.0007 formula XIV 2.7 0.2 formula XV 0.08 0.004 example 4  0.055 0.006example 5  0.005 0.008 example 6  0.055 0.095 example 7  0.006 0.050example 9  0.003 0.002 example 10 0.009 0.0035 example 11 0.012 0.006example 12 0.006 0.0085 example 13 0.004 0.006 example 14 0.005 0.01example 15 0.005 0.015 example 16 0.005 0.015 example 17 0.0075 0.003example 18 0.0025 0.005 example 19 0.0135 0.0065 example 20 0.008 0.0015example 21 0.009 0.0025 example 22 0.04 0.006 example 23 0.003 0.005example 24 0.004 0.007 example 25 0.0025 0.00375 example 26 0.00450.00085 example 27 0.02 0.003 example 28 0.03 0.0025 example 29 0.0250.003 example 30 4.55 1.05 example 31 0.3 0.2 example 32 0.03 0.1example 33 0.035 0.0045 example 34 0.005 0.004 example 35 0.003 0.0085example 36 0.0035 0.0085 example 37 0.0025 0.0045 example 38 0.00150.004 example 39 0.0035 0.0085 example 40 0.0025 0.0065 example 41 0.0010.002 example 42 0.001 0.001 example 43 0.001 0.0002 example 44 0.0090.0035 example 45 0.003 0.002 example 46 0.1 0.01 example 47 0.01 0.1

Example 2.2 HNE Inhibitory Activity In Vitro

Materials and Buffers

Human neutrophil elastase (cat. no. SE563) is purchased from ElastinProducts Company, Inc. (EPC, Owensville, USA). The dry powder(purity >95% stated by the supplier) was dissolved in 20 mM sodiumacetate buffer, pH 5.0, 50% (v/v) glycerol, and frozen at −80° C. inaliquots.

The fluorescence-quenched substrate(DABCYL-Ser-Glu-Val^Asn-Leu-Asp-Ala-Glu-Phe-EDANS, where ^ indicates thescissile bond, identified by MS analysis) was purchased from Bachem AG(Bubendorf, Switzerland), and kept as a 5 mM stock solution in DMSO at−20° C. All other chemicals were of analytical grade.

Enzymatic reactions were conducted in 100 mM Tris/HCl buffer at pH 7.5,containing 500 mM NaCl, and 0.05% (w/v) CHAPS.

All protein and peptide containing solutions are handled in siliconizedtubes (Life Systems Design, Merenschwand, Switzerland).

The compound solutions as well as the enzyme and the substrate solutionsare transferred to the 384-well plates (black Cliniplate; cat. no.95040020 Labsystems Oy, Finland) by means of a CyBi-Well 96-channelpipettor (CyBio AG, Jena, Germany).

Instrumentation for FI Measurements

For fluorescence intensity (FI) measurements an Ultra Evolution reader(TECAN, Maennedorf, Switzerland) is used. The instrument is equippedwith a combination of a 350 nm (20 nm bandwidth) and a 500 nm (25 nmbandwidth) bandpass filter for fluorescence excitation and emissionacquisition, respectively. To increase the signal:background ratio, anappropriate dichroic mirror is employed. The optical filters and thedichroic mirror are purchased from TECAN. The fluorophores in each wellare excited by three flashes per measurement.

The cyclic depsipeptides inhibited human neutrophile elastase with IC50values as indicated in table 11.

In addition the cyclic depsipeptides inhibited human chymotrypsin withan IC50 ranging from 0.001 μM to 0.02 μM.

The biological activity of the cyclic depsipeptide according to formula(VIII) was determined with kallikrein 7. This cyclic depsipeptide of theinvention inhibits human kallikrein 7 with an IC₅₀ of less than 3 nM.This cyclic depsipeptide inhibited human chymotrypsin and humanneutrophile elastase with an IC50 around 0.004 μM and around 0.0025 μM,respectively.

Example 3 Determination of Kallikrein-7 Inhibitory Activity In Vivo

A) Test on Recovery of Skin Barrier Disruption in Mice

Method: Skin barrier disruption was achieved in groups of hairless SKH1mice with repeated stripping of the skin with S-Sqame® skin samplingdisks. The procedure was completed when transepidermal water loss (TEWL)achieved ≧40 mg/cm²/h. TEWL was assessed with a Tewameter TM210 (CourageKhazaka, Cologne, Del.). Immediately after barrier disruption 30 μl thetest compound was applied at 10 mM concentration. Control animals weretreated similarly with the solvent (ethanol/propylene glycol, 3/7 (v/v))alone. TEWL was measured before, immediately after, and at 3 hrs afterbarrier disruption. In each animal, the percentage recovery wascalculated using the formula: (1−[TEWL at 3 hrs−base line TEWL]/[TEWLimmediately after stripping−base line TEWL])×100%.

Results:

A single application of the test compound (the Cyclic depsipeptideaccording to formula II) accelerated barrier repair by 57% compared torepair in mice treated with the solvent alone (p<0.05), Table 12.

TABLE 12 % Recovery in barrier disruption Mean Animals (SD values), n: 4animals per group) Treated with test compound 72.0 (9.1) (the Cyclicdepsipeptide according to formula II) at 10 mM Treated with solventalone 45.8 (8.0)B) Test on Anti-Inflammatory Activity in Murine Model of AllergicContact Dermatitis (ACD)

Method: Crl:NMRI mice were sensitized on the shaved abdomen with 50 μlof 2% oxazolone on day 1 and challenged with 10 μl oxazolone on theinner surface of the right ear on day 8. The unchallenged left earsserved as normal controls and dermatitis was evaluated from thedifference in auricular weight (taken as a measure of inflammatoryswelling) on day 9. The animals were treated topically with 10 μl testcompound or the solvent only 30 min after the challenge. The efficacy ofthe treatment was calculated as the percentage inhibition ofinflammatory auricular swelling relative to animals treated with thevehicle alone.

Results: A single application of the test compound (the Cyclicdepsipeptide according to formula II) inhibited inflammatory swelling inACD by 40% at 10 mM) and by 46% at 30 mM concentration (p<0.001 vssolvent-treated animals (Table 13/14).

TABLE 13 Δ Auricular % Inhibition of weights Mean inflammatory (SD), n:8 swelling Animals animals per group Mean ± SE Treated with testcompound 15.3 (5.4) 46 ± 7.5 (the Cyclic depsipeptide according toformula II) at 30 mM Treated with test compound 17.0 (4.8) 40 ± 6.9 (theCyclic depsipeptide according to formula II) at 10 mM Treated withsolvent⁺ alone 28.1 (4.6) — ⁺ mixture ofdimethylacetamide/ethanol/acetone (1/212)

TABLE 14 % Inhibition of % Inhibition of inflammatory inflammatoryswelling swelling concentration of concentration of test compound testcompound Compound according 30 mM 10 mM to formula example 9  38 42example 10 25 22 example 23 40 48 example 25 38 39 example 43 55 42

C) Test on Anti-Inflammatory Activity in Swine Model of Allergic ContactDermatitis (ACD)

Eight days before the elicitation of the ACD, 500 μl of 10%2,4-dinitrofluorobenzene (DNFB, dissolved in DMSO/acetone/olive oil[1/5/3, v/v/v]) were applied epicutaneously in divided volumes onto thebasis of both ears and onto both groins (100 μl/site) for sensitization.The challenge reactions were elicited with 15 μl of DNFB (1.0%) oncontralateral test sites (each 7 cm² in size) of the shaved dorsolateralback. For treatment, the test compound and the placebo (solvent only)were applied contralaterally to 2 test sites in each animal 0.5 and 6hrs after the challenge. The test sites were clinically examined 24 hrsafter the challenge when inflammation peaked. The changes were scored ona scale from 0 to 4 (Table 15), allowing a combined maximal score of 12per designated site. Skin reddening was measured reflectometricallyusing a* values.

TABLE 15 Scoring of clinical signs of test sites affected with ACDErythema/ Erythema/ Score Intensity Extent Induration 0 absent absentabsent 1 scarcely visible small spotted scarcely palpable 2 mild largespotted mild hardening 3 pronounced confluent pronounced hardening 4severe (or livid homogenous pronounced and discoloring) redness elevatedhardening

Results: Treatment of test sites affected with ACD twice with a 1%solution of the test compound (the Cyclic depsipeptide according toformula II) inhibited clinical inflammatory changes by 30% (p<0.01) andmeasured skin redness by 27% (p<0.05) (Table 16)

TABLE 16 Clinical score A* value Test sites (Mean, SD, n: 8+) (Mean, SD,n: 8+) Treated with 1% test compound 5.1 (1.7) 8.6 (1.4) (the Cyclicdepsipeptide according to formula II) Treated with placebo (solvent) 7.2(1.9) 12.0 (2.5) Inhibition vs placebo-treated sites 29.9 (11.7) 27.0(2.5) +2 test sites each in 4 animals

Example 4 Derivatisation of a Cyclic Depsipeptide of the Invention

a) One-step procedure: To a solution of 20 mg of cyclic depsipeptideaccording to formula (II) and 0.027 mL triethylsilane in 2 mL ofdichloromethane/acetonitrile (1:1) at −50° C. 0.014 mL of borontrifluoride etherate were slowly added. The reaction mixture was allowedto warm up to −5° C. and kept at this temperature for additional 30minutes, poured into a saturated NaHCO₃ solution, and was extracted withEtOAc. The organic layer was dried over sodium sulfate and the solventwas removed in vacuo. Purification of the residue obtained byHPLC(XTerra [5 cm]; acetonitrile/ammonium carbonate buffer pH10gradient) provided 9.8 mg of a derivative of the cyclic depsipeptideaccording to formula (II) wherein the Ahp has been converted into3-amino-piperidin-2-one. ESI MS: 935.36 [M+Na]⁺.b) Two-step procedure: To a solution of 1 g of cyclic depsipeptideaccording to formula (II) in 300 mL of dichloromethane/acetonitrile(1:1) at −50° C. 0.68 mL of boron trifluoride etherate were slowlyadded. The reaction mixture was allowed to warm up to −20° C. Thenadditional 0.68 mL of boron trifluoride etherate were slowly added thereaction mixture kept at this temperature until no more startingmaterial could be observed (HPLC). Then the reaction mixture was pouredinto a saturated NaHCO₃ solution, and was extracted with EtOAc. Theorganic layer was dried over sodium sulfate and the solvent was removedin vacuo providing a derivative of the cyclic depsipeptide according toformula (II) wherein the Ahp has been converted into3-amino-3,4-dihydro-1H-pyridin-2-one. ESI MS: 933.28 [M+Na]⁺.

The crude material was dissolved in 400 mL of 2-propanol, 115 mg of Pd/C(10%) were added and the mixture was hydrogenated under atmosphericpressure until the starting material was consumed (HPLC). The residueobtained was purified by chromatography (SiO₂; cHex/EtOAc (1:1)+10%MeOH) providing 684 mg of the cyclic depsipeptide according to formula(II) wherein the Ahp has been converted into 3-amino-piperidine-2-one.

Example 5 Derivatisation of a Cyclic Depsipeptide of the Invention

To a solution of 75 mg (0.081 mmol) of cyclic depsipeptide according toformula (II) in 5 mL of 1-PrOH 30 μL of sulfuric acid were added and thereaction mixture was stirred at r.t. for 48 hours. For workup thereaction mixture was diluted with dichloromethane and washed with sat.bicarbonate solution. After drying of the organic layer over sodiumsulfate the solvent was removed and the residue obtained purified bychromatography on silica gel (cHex/EtOAc (1:1)+10% MeOH). Yield: 65 mg(83%) of a derivative of the cyclic depsipeptide according to formula(II) wherein the Ahp has been converted into3-amino-6-propoxy-piperidin-2-one. ESI MS: 993.37 [M+Na]⁺.

Similarly, treatment with the corresponding alcohol provided thefollowing compounds:

TABLE 17 Example R ESI MS [M + Na]⁺ 6 1-octyl 1063.41 72,2,2-trifluoroethyl 1033.30 8 2-propyl 993.43 9 benzyl 1041.16 10 ethyl979.22 11 1-butyl 1007.29 12 isobutyl 1007.35 13 2-methoxyethyl 1009.3114 2-hydroxyethyl 995.28 15 2-(2-hydroxyethoxypethyl 1039.31 162-(2-methoxyethoxy)ethyl 1053.33 17 methyl 965.27 18 propargyl 989.21

Simultaneously, compounds of the following type were obtained under thesame conditions:

TABLE 18 Example R1, R2 ESI MS [M + Na]⁺ 19 1-propyl 1136.33 20 methyl980.21

Example 21 Derivatisation of a Cyclic Depsipeptide of the Invention

A solution of 25 mg (0.027 mmol) of cyclic depsipeptide according toformula (II) in 2 mL of dichloromethane was cooled to 0° C. Then DIPEAand trifluoroacetic acid anhydride (TFAA) was added. The reactionmixture was slowly warmed up to room temperature and stirred foradditional 4 hours. For workup the reaction mixture was diluted withdichloromethane and washed with hydrochloric acid and sat. bicarbonatesolution. After drying over sodium sulfate the solvent was removed andthe residue obtained purified by chromatography on silica gel(cHex/EtOAc (1:1)+10% MeOH). Yield: 14 mg (57%) of a derivative of thecyclic depsipeptide according to formula (II) wherein the amide in thesidechain of A1 has been converted into a nitrile. ESI MS: 933.30[M+Na]⁺.

Similarly, compounds of the following type was obtained:

TABLE 19 Example R ESI MS [M + Na]⁺ 22 H 917.30 23 ethyl 961.20 241-propyl 975.29 25 benzyl 1023.14

Example 26 Derivatisation of a Cyclic Depsipeptide of the Invention

A solution of 25 mg (0.027 mmol) of a cyclic depsipeptide according toformula (II) in 2 mL of dichloromethane (MC) was cooled to 0° C. Then 24μL of DIPEA and 14 μL of hexyl chloroformate was slowly added. Thereaction mixture was allowed to warm up to rt and stirred for additional4 hours. For workup the reaction mixture was diluted withdichloromethane and washed with hydrochloric acid and sat. bicarbonatesolution, and brine. After drying over sodium sulfate the solvent wasremoved and the residue obtained purified by chromatography on silicagel (cHex/EtOAc (1:1)+10% MeOH). Yield: 20 mg (70%) of a derivative ofthe cyclic depsipeptide according to formula (II) wherein the phenolmoiety of A6 has been transformed into the corresponding carbonic acidhexyl ester. ESI MS: 1079.41 [M+Na]⁺.

Analogously, using a compound described in Example 4 as startingmaterial the following compounds were obtained:

TABLE 20 Ex- ESI MS ample R [M + Na]⁺ 27 isobutyl 1035.38 282-methoxyethyl 1037.36 29 ethyl 1007.35 30

1347.63 31 1-octyl 1091.42

Example 32 Derivatisation of a Cyclic Depsipeptide of the Invention

To a mixture of 200 mg (0.21 mmol) of cyclic depsipeptide accordingExample 5, 57.5 mg (0.41 mmol) of K₂CO₃ in 2 mL of dry acetone 60.5 mg(0.31 mmol) of (E)-3-phenyl-2-propenyl bromide was added and treatedwith ultrasound overnight (temperature raises to about 50° C.). Forworkup the reaction mixture was diluted with dichloromethane and washedwith water. After drying of the organic layer over sodium sulfate thesolvent was removed and the residue obtained purified by HPLC (15 cmZorbax; acetonitrile/aqu. NH₄OAc buffer: 20→95%). Yield: 100 mg (45%) ofa derivative of cyclic depsipeptide according Example 5 featuringO-((E)-3-phenyl-2-propen-1-yl)-L-tyrosine in A6. ESI MS: 1109.37[M+Na]⁺.

Analogously, treatment of cyclic depsipeptides according Example 4 or 5with the appropriate alkylating agent provide the following compounds:

TABLE 21 Example R1 R2 ESI MS [M + Na]⁺ 33 H t-buthoxycarbonylnethyl1049.33 34 propoxy t-buthoxycarbonylmethyl 1107.33 35 propoxy1-(E)-pent-2-enyl 1061.36 36 propoxy 1-(E)-4,4,4-trifluoro-but-2-enyl1101.25 37 propoxy methyl 1007.29 38 propoxy 3-methyl-but-2-enyl 1061.3639 propoxy benzyl 1083.38 40 propoxy allyl 1033.37 41 propoxy propargyl1031.25

Example 42 Derivatisation of a Cyclic Depsipeptide of the Invention

To a mixture of 200 mg (0.21 mmol) of cyclic depsipeptide accordingExample 5, 46.5 mg (0.31 mmol) of sodium iodide, and 57.5 mg (0.41 mmol)of K₂CO₃ in 2 mL of dry acetone 44 mg (0.31 mmol) of3-(chloromethyl)-1,5-dimethyl-1H-pyrazole was added and treated withultrasound overnight (temperature raises to about 50° C.). For workupthe reaction mixture was diluted with dichloromethane and washed withwater. After drying of the organic layer over sodium sulfate the solventwas removed and the residue obtained purified by HPLC (15 cm Zorbax;acetonitrile/aqu. NH₄OAc buffer: 20→95%). Yield: 90 mg (40.5%) of aderivative of cyclic depsipeptide according Example 5 featuringO-(1,5-dimethyl-1H-pyrazol-3-yl)methyl-L-tyrosine in A6. ESI MS: 1101.39[M+Na]⁺.

Example 43

Analogously, treatment of cyclic depsipeptide of cyclic depsipeptideaccording to formula (II) with 3-(chloromethyl)-1,5-dimethyl-1H-pyrazoleprovides a compound featuring0-(1,5-dimethyl-1H-pyrazol-3-yl)methyl-L-tyrosine in A6. ESI MS: 1059.19[M+Na]⁺.

Example 44 Derivatisation of a Cyclic Depsipeptide of the Invention

To a solution of 17 mg (0.0165 mmol) of cyclic depsipeptide accordingExample 33 in 2 mL of dichloromethane 845 μL of trifluoroacetic acid wasadded and stirred at r.t. for 4 hours. The reaction mixture was dilutedwith toluene and the solvent was removed in vacuo providing 22.5 mg ofthe corresponding crude acid.

20 mg of the aforementioned acid, 6.81 mg (0.031 mmol) of8-amino-3,6-dioxaoctanoic acid tert-butylester, and 15.8 mg (0.041 mmol)of HATU were dissolved in 2 mL of dry DMF and 11 μL of DIEPA were addedand stirred at r.t. overnight. For workup, the reaction mixture wasdiluted with EtOAc and washed with sat. NaHSO₄ and NaHCO₃ solutions andbrine. After drying of the organic layer over sodium sulfate the solventwas removed and the residue obtained purified by HPLC (15 cm Zorbax;acetonitrile/aqu. NH₄OAc buffer: 20→95%). Yield: 7 mg (29%) of the titlecompound. ESI MS: 1194.32 [M+Na]⁺.

8-Amino-3,6-dioxaoctanoic acid tert-butylester

100 mg (0.226 mmol) of8-(9-Fluorenylmethoxycarbonylamino)-3,6-dioxaoctanoic acidtert-butylester in 1 mL of dry DMF were treated with piperidine (89.5μL; 0.862 mmol) at r.t. for 3 hours. The solvent was evaporated and theresidue purified by chromatography on silica gel with a gradientcHex→EtOAc→EtOAc/MeOH (1:1)+3%. MeOH. Yield: 12 mg (24%) of the titlecompound. ESI MS: 220.08 [M+H]⁺.

8-(9-Fluorenylmethoxycarbonylamino)-3,6-dioxaoctanoic acidtert-butylester

A solution of 150 mg (0.389 mmol) of8-(9-Fluorenylmethoxycarbonylamino)-3,6-dioxaoctanoic acid, 546 mg (9.73mmol) isobutylene, and 4.3 μL of 95-98% H₂SO₄ was stirred at r.t. for 3days. For workup the reaction solution was diluted with dichloromethaneand washed with sat. bicarbonate solution. After drying over sodiumsulfate the solvent was removed and the residue obtained purified bychromatography on silica gel with a cHex/EtOAc gradient providing 145 mg(84.4%) of the title compound. ESI MS: 464.12 [M+Na]⁺.

Example 45 Derivatisation of a Cyclic Depsipeptide of the Invention

To a mixture of 101 mg (0.1 mmol) of a compound of Example 41 and 40 mgCuI in 11 ml of toluene/DMF (10:1) 50 L of DIEPA and 1 mL of a 1Msolution of 1-azido-2-[2-(2-azido-ethoxy)-ethoxy]-ethane was added andstirred at 45° C. for 6 hours. Then the reaction mixture was washed witha sat. NaH₂PO₄ solution, dried over sodium sulfate, and the solvent wasevaporated. The residue obtained was purified by chromatography (SiO₂;cHex/EtOAc (1:1)+20% MeOH) providing 14 mg (11.7%) of the titlecompound. ESI MS: 1231.34 [M+Na]⁺.

Example 46 Derivatisation of a Cyclic Depsipeptide of the Invention

A solution of 25 mg of depsipeptide according to formula (II) in 25 mLwater was stirred at room temperature. In this solution an additionalpeak was observed in HPLC analysis, which forms an equilibrium with thedepsipeptide according to formula (II). After 20 days the solution wasdried using lyophilization and the additional peak was isolated usingreversed phase chromatography as described in example 1. This provided0.75 mg cyclic depsipeptide according to example 46, wherein Ahp hasbeen converted into 5-hydroxyproline.

ESI-MS: pos. mode: m/z=951.5 (M+Na), neg. Mode: m/z=927.4 (M−H);monoisotopic MW 928.5, C₄₆H₇₂N₈O₁₂

¹H NMR (600 MHz) d₅-DMSO δ_(H): 0.00 (1H, m), 0.50 (3H, t, J=7.3 Hz),0.71 (3H, m), 0.76 (3H, t, J=7.0 Hz), 0.76 (3H, d, J=7.3 Hz), 0.85 (611,d, J=6.6 Hz), 0.88 (1H, m), 1.00 (1H, m), 1.03 (3H, d, J=6.6 Hz), 1.06(6H, d, J=6.6 Hz), 1.10 (1H, m), 1.16 (1H, m), 1.26 (1H, m), 1.36 (1H,m), 1.43 (1H, m), 1.51 (2H, m), 1.79 (1H, m), 1.83 (1H, m), 1.97 (1H,m), 1.99 (1H, m), 2.17 (2H, t, J=7.7 Hz), 2.39 (1H, m), 2.48 (1H, m),2.67 (3H, s), 2.78 (1H, m), 3.43 (1H, m), 4.32 (1H, m), 4.33 (1H, m),4.45 (1H, m), 4.48 (1H, m), 4.58 (1H, m), 4.61 (1H, m), 4.67 (1H, m),5.09 (1H, m), 5.47 (1H, m), 6.66 (2H, d, J=8.1 Hz), 6.74 (1H, s, broad),7.03 (2H, d, J=8.1 Hz), 7.31 (1H, s, broad), 7.33 (1H, d, J=9.5 Hz),8.04 (1H, d, J=9.5 Hz), 8.17 (1H, d, J=8.1 Hz), 8.23 (1H, d, J=2.9 Hz),8.43 (1H, d, J=9.5 Hz), 9.25 (1H, s, broad), (OH group of hydroxyprolinenot visible).

Example 47 Derivatisation of a Cyclic Depsipeptide of the Invention

A solution of 100 mg of depsipeptide according to formula (II) in 25 mL0.5 N HCl was stirred at 50° C. for 24 h. For workup the pH of thereaction mixture was adjusted to pH 7 with 5 N NaOH and was extractedwith ethyl acetate. The organic layer was dried over sodium sulfate andthe solvent was removed in vacuo. The residue obtained was purified byreversed phase chromatography (same conditions as described example 1)providing 17 mg of cyclic depsipeptide according to example 47, whereinglutamine in A1 has been replaced by glutamic acid.

ESI-MS: pos. mode: m/z=952.8 (M+Na), neg. Mode: m/z=928.5 (M−H);monoisotopic MW 929.5, C₄₆H₇₁N₇O₁₃

¹H NMR (600 MHz, d₆-DMSO) δ_(H): −0.11 (3H, d, J=6.6 Hz), 0.64 (4H, m),0.77 (3H, d, J=6.6 Hz), 0.81 (3H, t, J=7.3 Hz), 0.84 (3H, d, J=6.6 Hz),0.88 (3H, d, J=6.6 Hz), 1.02 (6H, m), 1.05 (1H, m), 1.10 (1H, m), 1.19(3H, d, J=5.9 Hz), 1.24 (1H, m), 1.40 (1H, m), 1.51 (1H, m), 1.75 (1H,m), 1.78 (5H, m), 1.85 (2H, m), 2.10 (2H, m), 2.45 (1H, m), 2.60 (1H,m), 2.67 (1H, m), 2.71 (3H, s), 3.20 (1H, m), 4.28 (1H, m), 4.29 (1H,m), 4.44 (2H, m), 4.63 (1H, d, J=9.5 Hz), 4.69 (1H, m), 4.93 (1H, m),5.04 (1H, m), 5.47 (1H, m), 6.24 (1H, s, broad), 6.64 (2H, d, J=8.8),6.99 (2H, d, J=8.8 Hz), 7.37 (1H, d, J=9.5 Hz), 7.69 (1H, d, J=9.5 Hz),7.80 (1H, d, J=9.5 Hz), 8.51 (1H, d, J=8.8 Hz), 8.57 (1H, d, J=5.1 Hz),OH group of Tyrosine and glutamic acid not visible)

REFERENCES

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The invention claimed is:
 1. A method of treating a subject sufferingfrom psoriasis comprising administering to said subject atherapeutically effective amount of a cyclic depsipeptide, having thestructure of formula (I):

wherein an ester bond is formed between the carboxy group of A₇ and thehydroxy group of A₂, wherein X and A₁ are each independently optional,and wherein X is H or an amino group modifying moiety selected from anaryl carbonyl residue or from an acyl residue, A₁ is glutamine,ornithine, glutamic acid or a derivative thereof selected from glutamicnitrile, glutamic acid C₁₋₁₂alkyl ester or glutamic acid C₆₋₂₄arylester; A₂ is threonine or serine, A₃ is leucine, A₄ is Ahp,3-amino-piperidine-2-one, dehydro-AHP, Ahp-I, Ahp-II, proline,5-hydroxy-proline, wherein the point of fusion (with A₃ and A₅) are atthe nitrogen atom and the carboxyl oxygen (by virtue of the replacementof a hydrogen atom by a bond) of the proline, and 5-hydroxyproline,wherein Ahp, 3-amino-piperidine-2-one, dehydro-AHP, Ahp-I, and Ahp-II,are as defined below, and wherein the points of fusion (with A₃ and A₅)are at the nitrogen atoms of the said compounds (by virtue of thereplacement of a hydrogen atom by a bond):

wherein X is O or a bond, and R is an organic moiety or is a radicalselected from the group consisting of hydrogen, (C₁₋₁₂)alkyl,(C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl, halo(C₂₋₁₂) alkenyl,halo(C₂₋₁₂)alkynyl, (C₁₋₁₂)alkoxycarbonyl,(C₁₋₁₂)alkoxy-carbonyl(C₁₋₁₂)alkyl, (C₁₋₁₂) alkylaminocarbonyl,unsubstituted or further substituted by aryl, arylalkyl, arylalkenyl orarylalkynyl, heterocyclyl and heterocyclylalkyl, A₅ is isoleucine,phenylalanine or valine, A₆ is tyrosine, N-Me-tyrosine or a derivativethereof selected from N-Me-tyrosine where the OH group of the tyrosineor N-Me-tyrosine is OR, wherein R is selected from the group consistingof (C₁₋₁₂)alkyl, (C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl,halo(C₂₋₁₂)alkenyl, halo(C₂₋₁₂)alkynyl, (C₁₋₁₂)alkoxycarbonyl,(C₁₋₁₂)alkoxy-carbonyl(C₁₋₁₂)alkyl, (C₁₋₁₂) alkylaminocarbonyl, whereinthe alkyl, alkenyl and alkinyl moieties of groups R can be unsubstitutedor further substituted by aryl, aryl(C₁₋₆)alkyl, aryl(C₂₋₁₂)alkenyl oraryl(C₂₋₁₂) alkynyl, heterocyclyl and heterocyclyl(C₁₋₁₂)alkyl, A₇ isleucine, isoleucine or valine, or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1 wherein X is CH₃CH₂CH(CH₃)CO,(CH₃)₂CHCH₂CO, (CH₃)₂CHCO, CH₃CO or Phenyl-CO, or a pharmaceuticallyacceptable salt thereof.
 3. The method of claim 1 wherein the nitrogenatom of the amide bond between A5 and A6 is substituted with a methyland the OH group of the tyrosine is OR, wherein R is selected from thegroup consisting of hydrogen, (C₁₋₁₂)alkyl, (C₂₋₁₂) alkenyl,(C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl, halo(C₂₋₁₂)alkenyl,halo(C₂₋₁₂)alkynyl, (C₁₋₁₂)alkoxycarbonyl,(C₁₋₁₂)alkoxy-carbonyl(C₁₋₁₂)alkyl, (C₁₋₁₂)alkylaminocarbonyl,unsubstituted or further substituted by aryl, arylalkyl, arylalkenyl orarylalkynyl, heterocyclyl and heterocyclylalkyl, or a pharmaceuticallyacceptable salt thereof.
 4. The method of claim 1 wherein A4 is the Ahpderivative 3-amino-piperidin-2-one, Ahp-I or Ahp-II, wherein R isselected from the group consisting of of (C₁₋₁₂)alkyl, (C₂₋₁₂)al-kenyl,(C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl, (C₁₋₁₂)alkoxy(C₁₋₁₂)alkyl,(C₁₋₁₂)alkoxy(C₁₋₁₂)alkoxy(C₁₋₁₂)alkyl, hydroxy(C₁₋₁₂)alkyl, phenyl andphenyl(C₁₋₆)alkyl, or a pharmaceutically acceptable salt thereof.
 5. Themethod of claim 1 wherein the acyl residue X is CH₃CH₂CH(CH₃)CO or(CH₃)₂CHCO, A₁ is glutamine, glutamic acid, or a derivative thereofselected from glutamic nitrile, glutamic acid C₁₋₁₂alkyl ester orglutamic acid C₆₋₂₄aryl ester, A₂ is threonine, A₃ is leucine, A₄ isAhp, 3-amino-piperidine-2-one, proline, 5-hydroxy-proline, A₅ isisoleucine, A₆ is tyrosine, N-Me-tyrosine or a derivative thereofselected from N-Me-tyrosine where the OH group of the tyrosine orN-Me-tyrosine is OR, wherein R is selected from the group consisting of(C₁₋₁₂)alkyl, (C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl,halo(C₂₋₁₂)alkenyl, halo(C₂₋₁₂)alkynyl, (C₁₋₁₂)alkoxycarbonyl,(C₁₋₁₂)alkoxy-carbonyl(C₁₋₁₂)alkyl, (C₁₋₁₂) alkylaminocarbonyl, whereinthe alkyl, alkenyl and alkinyl moieties of groups R can be unsubstitutedor further substituted by aryl, aryl(C₁₋₆)alkyl, aryl(C₂₋₁₂)alkenyl oraryl(C₂₋₁₂) alkynyl, heterocyclyl and heterocyclyl(C₁₋₁₂)alkyl, A₇ isisoleucine or valine, or a pharmaceutically acceptable salt thereof. 6.The method of claim 1 wherein A₄ is Ahp, Ahp-I,3-amino-piperidine-2-one, proline, or 5-hydroxy-proline, or apharmaceutically acceptable salt thereof.
 7. The method of claim 1,wherein the cyclic depsipeptide is a compound in accordance withformulae A or B,

wherein X and A₁ are as defined in claim 1, and wherein R2 is methyl orhydrogen, R3 is the side chain of leucine, R5 is the side chain of theamino acid isoleucine or valine, in particular R5 stands for isoleucine,R6 is the side chain of tyrosine which is optionally derivatized on itshydroxyl group, wherein the hydroxyl group of the tyrosine is OR, andwherein R is selected from the group consisting of hydrogen,(C₁₋₁₂)alkyl, (C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl,halo(C₂₋₁₂) alkenyl, halo(C₂₋₁₂)alkynyl, (C₁₋₁₂)alkoxycarbonyl,(C₁₋₁₂)alkoxy-carbonyl(C₁₋₁₂)alkyl, (C₁₋₁₂) alkylaminocarbonyl,unsubstituted or further substituted by aryl, arylalkyl, arylalkenyl orarylalkynyl, heterocyclyl and heterocyclylalkyl, R7 is the side chain ofthe amino acid leucine, isoleucine or valine, Y is either hydrogen or amethyl, or a pharmaceutically acceptable salt thereof.
 8. The method ofclaim 1 wherein A1, A2, A3, A5, A6 and A7 are L-amino acids, or apharmaceutically acceptable salt thereof.
 9. The method of claim 1wherein A4 is 3S,6R Ahp, or a pharmaceutically acceptable salt thereof.10. The method of claim 1 wherein A1 is a glutamine, ornithine, or aglutamine derivative selected from glutamic nitrile, and glutamic acidester, or a pharmaceutically acceptable salt thereof.
 11. A process forproducing the cyclic depsipeptide of Formula I:

wherein the acyl residue X is CH₃CH₂CH(CH₃)CO or (CH₃)₂CHCO, A₁ isglutamine, glutamic acid, or a derivative thereof selected from glutamicnitrile, glutamic acid C₁₋₁₂alkyl ester or glutamic acid C₆₋₂₄arylester, A₂ is threonine, A₃ is leucine, A₄ is Ahp,3-amino-piperidine-2-one, proline, 5-hydroxy-proline, A₅ is isoleucine,A₆ is tyrosine, N-Me-tyrosine or a derivative thereof selected fromN-Me-tyrosine where the OH group of the tyrosine or N-Me-tyrosine is OR,wherein R is selected from the group consisting of (C₁₋₁₂)alkyl,(C₂₋₁₂)alkenyl, (C₂₋₁₂)alkynyl, halo(C₁₋₁₂)alkyl, halo(C₂₋₁₂)alkenyl,halo(C₂₋₁₂)alkynyl, (C₁₋₁₂)alkoxycarbonyl,(C₁₋₁₂)alkoxy-carbonyl(C₁₋₁₂)alkyl, (C₁₋₁₂) alkylaminocarbonyl, whereinthe alkyl, alkenyl and alkinyl moieties of groups R can be unsubstitutedor further substituted by aryl, aryl(C₁₋₆)alkyl, aryl(C₂₋₁₂)alkenyl oraryl(C₂₋₁₂) alkynyl, heterocyclyl and heterocyclyl(C₁₋₁₂)alkyl, A₇ isisoleucine or valine; or a pharmaceutically acceptable salt thereof;comprising expressing the biosynthesis genes of a Chondromyces crocatusa(DSM 19329) in a heterologous microbial host strain for a time and underconditions effective to produce a first cyclic depsipeptide of FormulaI, and optionally converting said first cyclic depsipeptide to a secondcyclic depsipeptide of Formula I by means of one or more chemicalreactions.
 12. The method according to claim 1 wherein the cyclicdepsipeptide is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.
 13. The method accordingto claim 1 wherein the cyclic depsipeptide is a compound of formula(II):

or a pharmaceutically acceptable salt thereof.
 14. The method accordingto claim 12 wherein the cyclic depsipeptide is a compound of formula(III); or a pharmaceutically acceptable salt thereof.
 15. The methodaccording to claim 1 wherein the cyclic depsipeptide is a compound offormula (IV):

or a pharmaceutically acceptable salt thereof.
 16. The method accordingto claim 1 wherein the cyclic depsipeptide is a compound of formula (V):

or a pharmaceutically acceptable salt thereof.
 17. The method accordingto claim 1 wherein the cyclic depsipeptide is a compound of formula(XIV):

or a pharmaceutically acceptable salt thereof.